The Hidden!: CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel

CDC-006-00019, Revision: 05 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 07/13/2020
CDC 2019-Novel Coronavirus (2019-nCoV)
#WakeUp
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Real-Time RT-PCR Diagnostic Panel
For Emergency Use Only
Instructions for Use
Catalog # 2019-nCoVEUA-01
1000 reactions
For In-vitro Diagnostic (IVD) Use
Rx Only
https://www.fda.gov/media/134922/downloadCenters for Disease Control and Prevention
Division of Viral Diseases
1600 Clifton Rd NE
Atlanta GA 30329
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Table of Contents
Intended Use ............................................................................................................................. 2
Summary and Explanation....................................................................................................... 2
Principles of the Procedure ..................................................................................................... 3
Materials Required (Provided)................................................................................................. 5
Materials Required (But Not Provided)................................................................................... 6
Warnings and Precautions .................................................................................................... 10
Reagent Storage, Handling, and Stability............................................................................. 11
Specimen Collection, Handling, and Storage....................................................................... 12
Specimen Referral to CDC ..................................................................................................... 13
Reagent and Controls Preparation........................................................................................ 13
General Preparation ............................................................................................................... 14
Nucleic Acid Extraction.......................................................................................................... 14
Assay Set Up........................................................................................................................... 16
Create a Run Template on the Applied Biosystems 7500 Fast Dx Real-time PCR
Instrument (Required if no template exists)......................................................................... 20
Defining the Instrument Settings .......................................................................................... 26
Running a Test........................................................................................................................ 29
Interpretation of Results and Reporting............................................................................... 34
2019-nCoV rRT-PCR Diagnostic Panel Results Interpretation Guide ................................ 36
Quality Control........................................................................................................................ 37
Limitations .............................................................................................................................. 37
Conditions of Authorization for the Laboratory................................................................... 38
Performance Characteristics................................................................................................. 39
Disposal................................................................................................................................... 49
References .............................................................................................................................. 49
Revision History ..................................................................................................................... 50
Contact Information, Ordering, and Product Support......................................................... 50
Appendix A: Heat Treatment Alternative to Extraction ....................................................... 51
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Intended Use
The CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel is a real-time RT-PCR
test intended for the qualitative detection of nucleic acid from the 2019-nCoV in upper and lower
respiratory specimens (such as nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract
aspirates, bronchoalveolar lavage, and nasopharyngeal wash/aspirate or nasal aspirate) collected from
individuals who meet 2019-nCoV clinical and/or epidemiological criteria (for example, clinical signs and
symptoms associated with 2019-nCoV infection, contact with a probable or confirmed 2019-nCoV case,
history of travel to geographic locations where 2019-nCoV cases were detected, or other epidemiologic
links for which 2019-nCoV testing may be indicated as part of a public health investigation). Testing in
the United States is limited to laboratories certified under the Clinical Laboratory Improvement
Amendments of 1988 (CLIA), 42 U.S.C. § 263a, to perform high complexity tests.
Results are for the identification of 2019-nCoV RNA. The 2019-nCoV RNA is generally detectable in upper
and lower respiratory specimens during infection. Positive results are indicative of active infection with
2019-nCoV but do not rule out bacterial infection or co-infection with other viruses. The agent detected
may not be the definite cause of disease. Laboratories within the United States and its territories are
required to report all positive results to the appropriate public health authorities.
Negative results do not preclude 2019-nCoV infection and should not be used as the sole basis for
treatment or other patient management decisions. Negative results must be combined with clinical
observations, patient history, and epidemiological information.
Testing with the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel is intended for use by trained
laboratory personnel who are proficient in performing real-time RT-PCR assays. The CDC 2019-Novel
Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel is only for use under a Food and Drug
Administration’s Emergency Use Authorization.
Summary and Explanation
An outbreak of pneumonia of unknown etiology in Wuhan City, Hubei Province, China was initially
reported to WHO on December 31, 2019. Chinese authorities identified a novel coronavirus (2019-
nCoV), which has resulted in millions of confirmed human infections globally. Cases of asymptomatic
infection, mild illness, severe illness, and deaths have been reported.
The CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel is a molecular in vitro diagnostic test that aids in
the detection and diagnosis 2019-nCoV and is based on widely used nucleic acid amplification
technology. The product contains oligonucleotide primers and dual-labeled hydrolysis probes (TaqMan®)
and control material used in rRT-PCR for the in vitro qualitative detection of 2019-nCoV RNA in
respiratory specimens.
The term “qualified laboratories” refers to laboratories in which all users, analysts, and any person
reporting results from use of this device should be trained to perform and interpret the results from this
procedure by a competent instructor prior to use.
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Principles of the Procedure
The oligonucleotide primers and probes for detection of 2019-nCoV were selected from regions of the
virus nucleocapsid (N) gene. The panel is designed for specific detection of the 2019-nCoV (two
primer/probe sets). An additional primer/probe set to detect the human RNase P gene (RP) in control
samples and clinical specimens is also included in the panel.
RNA isolated and purified from upper and lower respiratory specimens is reverse transcribed to cDNA
and subsequently amplified in the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with SDS
version 1.4 software. In the process, the probe anneals to a specific target sequence located between
the forward and reverse primers. During the extension phase of the PCR cycle, the 5’ nuclease activity of
Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye,
generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from
their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at
each PCR cycle by Applied Biosystems 7500 Fast Dx Real-Time PCR System with SDS version 1.4 software.
Detection of viral RNA not only aids in the diagnosis of illness but also provides epidemiological and
surveillance information.
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Summary of Preparation and Testing Process
Resuspend
primer/probe mix,
aliquot and store at
≤ -20°C
Resuspend and
aliquot nCoVPC,
store at -70°C
Extract sample RNA
and HSC RNA
Prepare master mix
(15 µL)
Prepare rRT-PCR
plate (5 µL RNA)
Run assay on
ABI 7500Fast Dx
Analyze data
Report results
Upon receipt of
rRT-PCR Panel
reagents
Upon obtaining
sample
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Materials Required (Provided)
Note: CDC will maintain on its website a list of commercially available lots of primer and probe sets
and/or positive control materials that are acceptable alternatives to the CDC primer and probe set
and/or positive control included in the Diagnostic Panel. Only material distributed through the CDC
International Reagent Resource and specific lots of material posted to the CDC website are acceptable
for use with this assay under CDC’s Emergency Use Authorization.
This list of acceptable alternative lots of primer and probe materials and/or positive control materials
will be available at:
https://www.cdc.gov/coronavirus/2019-nCoV/lab/virus-requests.html
Primers and Probes:
Catalog #2019-nCoVEUA-01 Diagnostic Panel Box #1:
Reagent Label Part # Description Quantity /
Tube
Reactions /
Tube
2019-nCoV_N1 RV202001
RV202015 2019-nCoV_N1 Combined Primer/Probe Mix 22.5 nmol 1000
2019-nCoV_N2 RV202002
RV202016 2019-nCoV_N2 Combined Primer/Probe Mix 22.5 nmol 1000
RP RV202004
RV202018 Human RNase P Combined Primer/Probe Mix 22.5 nmol 1000
Positive Control (either of the following products are acceptable)
Catalog #2019-nCoVEUA-01 Diagnostic Panel Box #2:
Reagent
Label Part # Description Quantity Notes
nCoVPC RV202005
2019-nCoV Positive Control (nCoVPC)
For use as a positive control with the CDC 2019-
nCoV Real-Time RT-PCR Diagnostic Panel
procedure. The nCoVPC contains noninfectious
positive control material supplied in a dried state
and must be resuspended before use. nCoVPC
consists of in vitro transcribed RNA. nCoVPC will
yield a positive result with each assay in the
2019-nCoV Real-Time RT-PCR Diagnostic Panel
including RP.
4 tubes
Provides
(800) 5 µL
test reactions
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Catalog #VTC-04 CDC 2019-nCoV Positive Control (nCoVPC)
Reagent
Label Part # Description Quantity Notes
nCoVPC RV202005
2019-nCoV Positive Control (nCoVPC)
For use as a positive control with the CDC 2019-
nCoV Real-Time RT-PCR Diagnostic Panel
procedure. The nCoVPC contains noninfectious
positive control material supplied in a dried state
and must be resuspended before use. nCoVPC
consists of in vitro transcribed RNA. nCoVPC will
yield a positive result with each assay in the
2019-nCoV Real-Time RT-PCR Diagnostic Panel
including RP.
4 tubes
Provides
(800) 5 µL
test reactions
Materials Required (But Not Provided)
Human Specimen Control (HSC)
Description Quantity CDC Catalog No.
Manufactured by CDC. For use as a nucleic acid extraction procedural
control to demonstrate successful recovery of nucleic acid as well as
extraction reagent integrity. The HSC consists of noninfectious (betaPropiolactone treated) cultured human cell material supplied as a liquid
suspended in 0.01 M PBS at pH 7.2-7.4.
10 vials x 500uL KT0189
Acceptable alternatives to HSC:
• Negative human specimen material: Laboratories may prepare a volume of human specimen
material (e.g., human sera or pooled leftover negative respiratory specimens) to extract and
run alongside clinical samples as an extraction control. This material should be prepared in
sufficient volume to be used across multiple runs. Material should be tested prior to use as
the extraction control to ensure it generates the expected results for the HSC listed in these
instructions for use.
• Contrived human specimen material: Laboratories may prepare contrived human specimen
materials by suspending any human cell line (e.g., A549, Hela, or 293) in PBS. This material
should be prepared in sufficient volume to be used across multiple runs. Material should be
tested prior to use as the extraction control to ensure it generates the expected results for
the HSC listed in these instructions for use.
CDC will maintain on its website a list of commercially alternative extraction controls, if applicable,
that are acceptable for use with this assay under CDC’s Emergency Use Authorization, at:
https://www.cdc.gov/coronavirus/2019-nCoV/lab/virus-requests.html
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rRT-PCR Enzyme Mastermix Options
Reagent Quantity Catalog No.
Quantabio qScript XLT One-Step RT-qPCR ToughMix
100 x 20 μL rxns
(1 x 1 mL) 95132-100
2000 x 20 μL rxns
(1 x 20 mL) 95132-02K
500 x 20 μL rxns
(5 x 1 mL) 95132-500
Quantabio UltraPlex 1-Step ToughMix (4X)
100 x 20 µL rxns
(500 µL) 95166-100
500 x 20 μL rxns
(5 x 500 µL) 95166-500
1000 x 20 μL rxns
(1 x 5 mL) 95166-01K
Promega GoTaq® Probe 1- Step RT-qPCR System
200 x 20 μL rxns
(2 mL) A6120
1250 x 20 μL rxns
12.5 mL A6121
Thermofisher TaqPath™ 1-Step RT-qPCR Master Mix, CG
1000 reactions A15299
2000 reactions A15300
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RNA Extraction Options
For each of the kits listed below, CDC has confirmed that the external lysis buffer is effective for
inactivation of SARS-CoV-2.
Instrument/Manufacturer Extraction Kit Catalog No.
QIAGEN
2
QIAmp DSP Viral RNA Mini Kit 50 extractions (61904)
2
QIAamp Viral RNA Mini Kit 50 extractions (52904)
250 extractions (52906)
QIAGEN EZ1 Advanced XL
2
EZ1 DSP Virus Kit
48 extractions (62724)
Buffer AVL (19073 or 19089)
EZ1 Advanced XL DSP Virus Card (9018703)
2
EZ1 Virus Mini Kit v2.0
48 extractions (955134)
Buffer AVL (19073 or 19089)
EZ1 Advanced XL Virus Card v2.0 (9018708)
Roche MagNA Pure 24 2
MagNA Pure 24 Total NA
Isolation Kit
96 extractions (07 658 036 001)
External Lysis Buffer (06 374 913 001, 12 239 469
103, 03 246 779 001 or 03 246 752 001)
Roche MagNA Pure 96 2
DNA and Viral NA Small Volume
Kit
576 extractions (06 543 588 001)
External Lysis Buffer (06 374 913 001, 12 239 469
103, 03 246 779 001 or 03 246 752 001)
1
Roche MagNA Pure LC 2
Total Nucleic Acid Kit 192 extractions (03 038 505 001)
1
Roche MagNA Pure Compact 2
Nucleic Acid Isolation Kit I 32 extractions (03 730 964 001)
Promega Maxwell® RSC 48 3
Maxwell® RSC Viral Total
Nucleic Acid Purification Kit
48 extractions (AS1330)
144 extractions (ASB1330)
1
QIAGEN QIAcube
2
QIAmp DSP Viral RNA Mini Kit 50 extractions (61904)
2
QIAamp Viral RNA Mini Kit
50 extractions (52904)
250 extractions (52906)
1, 3bioMérieux NucliSENS®
easyMAG®
and
1, 3bioMérieux EMAG®
(Automated magnetic
extraction reagents sold
separately. Both instruments
use the same reagents and
disposables, with the
exception of tips.)
EasyMAG® Magnetic Silica (280133)
EasyMAG® Lysis Buffer (280134)
EasyMAG® Lysis Buffer, 2 mL (200292)
EasyMAG® Wash Buffers 1,2, and 3
(280130, 280131, 280132)
EasyMAG® Disposables (280135)
Biohit Pipette Tips (easyMAG® only) (280146)
EMAG®1000μL Tips (418922)
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Equivalence and performance of these extraction platforms for extraction of viral RNA were demonstrated with the CDC
Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (K190302). Performance characteristics of these extraction
platforms with 2019-nCoV (SARS CoV-2) have not been demonstrated.
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2 CDC has confirmed that the external lysis buffer used with this extraction method is effective for inactivation of SARSCoV-2. 3 CDC has compared the concentration of inactivating agent in the lysis buffer used with this extraction method and has
determined the concentration to be within the range of concentrations found effective in inactivation of SARS-CoV-2.
Alternative to Extraction:
If a laboratory cannot access adequate extraction reagents to support testing demand due to the
global shortage of reagents, CDC has evaluated a heat treatment procedure for upper respiratory
specimens using the Quantabio UltraPlex 1-Step ToughMix (4X), CG. Though performance was
comparable, this method has been evaluated with a limited number of clinical specimens and a
potential reduction in sensitivity due to carryover of inhibitory substances or RNA degradation cannot
be ruled out. It should only be used when a jurisdiction determines that the testing need is great
enough to justify the risk of a potential loss of sensitivity. Heat-treated specimens generating
inconclusive or invalid results should be extracted with an authorized extraction method prior to
retesting. Details and procedure for the heat treatment alternative to extraction may be found in
Appendix A.
Equipment and Consumables Required (But Not Provided)
 Vortex mixer
 Microcentrifuge
 Micropipettes (2 or 10 μL, 200 μL and 1000 μL)
 Multichannel micropipettes (5-50 μl)
 Racks for 1.5 mL microcentrifuge tubes
 2 x 96-well -20°C cold blocks
 7500 Fast Dx Real-Time PCR Systems with SDS 1.4 software (Applied Biosystems; catalog
#4406985 or #4406984)
 Extraction systems (instruments): QIAGEN EZ1 Advanced XL, QIAGEN QIAcube, Roche MagNA
Pure 24, Roche MagNA Pure 96, Promega Maxwell® RSC 48, Roche MagNA Pure LC, Roche
MagNA Pure Compact, bioMérieux easyMAG, and bioMérieux EMAG
 Molecular grade water, nuclease-free
 10% bleach (1:10 dilution of commercial 5.25-6.0% hypochlorite bleach)
 DNAZapTM (Ambion, cat. #AM9890) or equivalent
 RNase AWAY™ (Fisher Scientific; cat. #21-236-21) or equivalent
 Disposable powder-free gloves and surgical gowns
 Aerosol barrier pipette tips
 1.5 mL microcentrifuge tubes (DNase/RNase free)
 0.2 mL PCR reaction plates (Applied Biosystems; catalog #4346906 or #4366932)
 MicroAmp Optical 8-cap Strips (Applied Biosystems; catalog #4323032)
Qualifying Alternative Components:
If a laboratory modifies this test by using unauthorized, alternative components (e.g., extraction
methods or PCR instruments), the modified test is not authorized under this EUA. FDA’s Policy for
Diagnostic Tests for Coronavirus Disease-2019 during the Public Health Emergency, updated May 11,
2020, does not change this. As part of this policy, FDA does not intend to object when a laboratory
modifies an EUA-authorized test, which could include using unauthorized components, without
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obtaining an EUA or EUA amendment, where the modified test is validated using a bridging study to the
EUA-authorized test .
Warnings and Precautions
• For in vitro diagnostic use (IVD).
• This test has not been FDA cleared or approved; this test has been authorized by FDA under
an EUA for use by laboratories certified under CLIA, 42 U.S.C. § 263a, to perform high
complexity tests.
• This test has been authorized only for the detection of nucleic acid from SARSCoV-2, not
for any other viruses or pathogens.
• This test is only authorized for the duration of the declaration that circumstances exist
justifying the authorization of emergency use of in vitro diagnostic tests for detection
and/or diagnosis of COVID-19 under Section 564(b)(1) of the Act, 21 U.S.C. § 360bbb3(b)(1), unless the authorization is terminated or revoked sooner.
• Follow standard precautions. All patient specimens and positive controls should be considered
potentially infectious and handled accordingly.
• Do not eat, drink, smoke, apply cosmetics or handle contact lenses in areas where reagents and
human specimens are handled.
• Handle all specimens as if infectious using safe laboratory procedures. Refer to Interim
Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with 2019-
nCoV https://www.cdc.gov/coronavirus/2019-nCoV/lab-biosafety-guidelines.html.
• Specimen processing should be performed in accordance with national biological safety
regulations.
• If infection with 2019-nCoV is suspected based on current clinical and epidemiological screening
criteria recommended by public health authorities, specimens should be collected with
appropriate infection control precautions.
• Performance characteristics have been determined with human upper respiratory specimens and
lower respiratory tract specimens from human patients with signs and symptoms of respiratory
infection.
• Perform all manipulations of live virus samples within a Class II (or higher) biological safety
cabinet (BSC).
• Use personal protective equipment such as (but not limited to) gloves, eye protection, and lab
coats when handling kit reagents while performing this assay and handling materials including
samples, reagents, pipettes, and other equipment and reagents.
• Amplification technologies such as PCR are sensitive to accidental introduction of PCR product
from previous amplifications reactions. Incorrect results could occur if either the clinical
specimen or the real-time reagents used in the amplification step become contaminated by
accidental introduction of amplification product (amplicon). Workflow in the laboratory should
proceed in a unidirectional manner.
 Maintain separate areas for assay setup and handling of nucleic acids.
 Always check the expiration date prior to use. Do not use expired reagents. Do not
substitute or mix reagents from different kit lots or from other manufacturers.
 Change aerosol barrier pipette tips between all manual liquid transfers.
 During preparation of samples, compliance with good laboratory techniques is essential
to minimize the risk of cross-contamination between samples and the inadvertent
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introduction of nucleases into samples during and after the extraction procedure. Proper
aseptic technique should always be used when working with nucleic acids.
 Maintain separate, dedicated equipment (e.g., pipettes, microcentrifuges) and supplies
(e.g., microcentrifuge tubes, pipette tips) for assay setup and handling of extracted
nucleic acids.
 Wear a clean lab coat and powder-free disposable gloves (not previously worn) when
setting up assays.
 Change gloves between samples and whenever contamination is suspected.
 Keep reagent and reaction tubes capped or covered as much as possible.
 Primers, probes (including aliquots), and enzyme master mix must be thawed and
maintained on a cold block at all times during preparation and use.
 Work surfaces, pipettes, and centrifuges should be cleaned and decontaminated with
cleaning products such as 10% bleach, DNAZap™, or RNase AWAY™ to minimize risk of
nucleic acid contamination. Residual bleach should be removed using 70% ethanol.
• RNA should be maintained on a cold block or on ice during preparation and use to ensure
stability.
• Dispose of unused kit reagents and human specimens according to local, state, and federal
regulations.
Reagent Storage, Handling, and Stability
• Store all dried primers and probes and the positive control, nCoVPC, at 2-8°C until re-hydrated for
use. Store liquid HSC control materials at ≤ -20°C.
Note: Storage information is for CDC primer and probe materials obtained through the
International Reagent Resource. If using commercial primers and probes, please refer to the
manufacturer’s instructions for storage and handling.
• Always check the expiration date prior to use. Do not use expired reagents.
• Protect fluorogenic probes from light.
• Primers, probes (including aliquots), and enzyme master mix must be thawed and kept on a cold
block at all times during preparation and use.
• Do not refreeze probes.
• Controls and aliquots of controls must be thawed and kept on ice at all times during preparation
and use.
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Specimen Collection, Handling, and Storage
Inadequate or inappropriate specimen collection, storage, and transport are likely to yield false test
results. Training in specimen collection is highly recommended due to the importance of specimen
quality. CLSI MM13-A may be referenced as an appropriate resource.
 Collecting the Specimen
• Refer to Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from
Patients Under Investigation (PUIs) for 2019 Novel Coronavirus (2019-nCoV)
https://www.cdc.gov/coronavirus/2019-nCoV/guidelines-clinical-specimens.html
• Follow specimen collection device manufacturer instructions for proper collection methods.
• Swab specimens should be collected using only swabs with a synthetic tip, such as nylon or
Dacron®, and an aluminum or plastic shaft. Calcium alginate swabs are unacceptable and
cotton swabs with wooden shafts are not recommended. Place swabs immediately into
sterile tubes containing 1-3 ml of appropriate transport media, such as viral transport media
(VTM).
 Transporting Specimens
• Specimens must be packaged, shipped, and transported according to the current edition of
the International Air Transport Association (IATA) Dangerous Goods Regulation. Follow
shipping regulations for UN 3373 Biological Substance, Category B when sending potential
2019-nCoV specimens. Store specimens at 2-8°C and ship overnight to CDC on ice pack. If a
specimen is frozen at -70°C or lower, ship overnight to CDC on dry ice.
 Storing Specimens
• Specimens can be stored at 2-8oC for up to 72 hours after collection.
• If a delay in extraction is expected, store specimens at -70oC or lower.
• Extracted nucleic acid should be stored at -70oC or lower.
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Specimen Referral to CDC
For state and local public health laboratories:
• Ship all specimens overnight to CDC.
• Ship frozen specimens on dry ice and non-frozen specimens on cold packs.
• Refer to the International Air Transport Association (IATA - www.iata.org) for requirements
for shipment of human or potentially infectious biological specimens. Follow shipping
regulations for UN 3373 Biological Substance, Category B when sending potential 2019-nCoV
specimens.
• Prior to shipping, notify CDC Division of Viral Diseases (see contact information below) that
you are sending specimens.
• Send all samples to the following recipient:
Centers for Disease Control and Prevention
c/o STATT
Attention: Unit 66
1600 Clifton Rd., Atlanta, GA 30329-4027
Phone: (404) 639-3931
The emergency contact number for CDC Emergency Operations Center (EOC) is
770-488-7100.
All other laboratories that are CLIA certified and meet requirements to perform high complexity
testing:
• Please notify your state and/or local public health laboratory for specimen referral and
confirmatory testing guidance.
Reagent and Controls Preparation
NOTE: Storage information is for materials obtained through the CDC International Regent Resource.
If using commercial products for testing, please refer to the manufacturer’s instructions for storage,
handling, and preparation instructions.
Primer and Probe Preparation:
1) Upon receipt, store dried primers and probes at 2-8°C.
2) Precautions: These reagents should only be handled in a clean area and stored at appropriate
temperatures (see below) in the dark. Freeze-thaw cycles should be avoided. Maintain cold
when thawed.
3) Using aseptic technique, suspend dried reagents in 1.5 mL of nuclease-free water and allow
to rehydrate for 15 min at room temperature in the dark.
4) Mix gently and aliquot primers/probe in 300 μL volumes into 5 pre-labeled tubes. Store a
single, working aliquot of primers/probes at 2-8oC in the dark. Store remaining aliquots at ≤ -
20oC in a non-frost-free freezer. Do not refreeze thawed aliquots (stable for up to 4 months at
2-8oC).
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2019-nCoV Positive Control (nCoVPC) Preparation:
1) Precautions: This reagent should be handled with caution in a dedicated nucleic acid handling
area to prevent possible contamination. Freeze-thaw cycles should be avoided. Maintain on
ice when thawed.
2) Resuspend dried reagent in each tube in 1 mL of nuclease-free water to achieve the proper
concentration. Make single use aliquots (approximately 30 μL) and store at ≤ -70oC.
3) Thaw a single aliquot of diluted positive control for each experiment and hold on ice until
adding to plate. Discard any unused portion of the aliquot.
Human Specimen Control (HSC) (not provided)
1) Human Specimen Control (HSC) or one of the listed acceptable alternative extraction controls
must be extracted and processed with each specimen extraction run.
2) Refer to the Human Specimen Control (HSC) package insert for instructions for use.
No Template Control (NTC) (not provided)
1) Sterile, nuclease-free water
2) Aliquot in small volumes
3) Used to check for contamination during specimen extraction and/or plate set-up
General Preparation
Equipment Preparation
Clean and decontaminate all work surfaces, pipettes, centrifuges, and other equipment prior to use.
Decontamination agents should be used including 10% bleach, 70% ethanol, and DNAzap™, or RNase
AWAY™ to minimize the risk of nucleic acid contamination.
Nucleic Acid Extraction
Performance of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel is dependent upon the amount
and quality of template RNA purified from human specimens. The following commercially available
RNA extraction kits and procedures have been qualified and validated for recovery and purity of RNA
for use with the panel:
Qiagen QIAamp® DSP Viral RNA Mini Kit or QIAamp® Viral RNA Mini Kit
Recommendation(s): Utilize 100 μL of sample and elute with 100 μL of buffer or utilize 140 μL of
sample and elute with 140 μL of buffer.
Qiagen EZ1 Advanced XL
Kit: Qiagen EZ1 DSP Virus Kit and Buffer AVL (supplied separately) for offboard lysis
Card: EZ1 Advanced XL DSP Virus Card
Recommendation(s): Add 120 μL of sample to 280 μL of pre-aliquoted Buffer AVL (total input sample
volume is 400 μL). Proceed with the extraction on the EZ1 Advanced XL. Elution volume is 120 μL.
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Kit: Qiagen EZ1 Virus Mini Kit v2.0 and Buffer AVL (supplied separately) for offboard lysis
Card: EZ1 Advanced XL Virus Card v2.0
Recommendation(s): Add 120 μL of sample to 280 μL of pre-aliquoted Buffer AVL (total input sample
volume is 400 μL). Proceed with the extraction on the EZ1 Advanced XL. Elution volume is 120 μL.
Roche MagNA Pure 96
Kit: Roche MagNA Pure 96 DNA and Viral NA Small Volume Kit
Protocol: Viral NA Plasma Ext LysExt Lys SV 4.0 Protocol or Viral NA Plasma Ext Lys SV Protocol
Recommendation(s): Add 100 μL of sample to 350 μL of pre-aliquoted External Lysis Buffer (supplied
separately) (total input sample volume is 450 μL). Proceed with the extraction on the MagNA Pure 96.
(Internal Control = None). Elution volume is 100 μL.
Roche MagNA Pure 24
Kit: Roche MagNA Pure 24 Total NA Isolation Kit
Protocol: Pathogen 1000 2.0 Protocol
Recommendation(s): Add 100 µL of sample to 400 µL of pre-aliquoted External Lysis Buffer (supplied
separately) (total input sample volume is 500 µL). Proceed with the extraction on the MagNA Pure 24.
(Internal Control = None). Elution volume is 100 µL.
Promega Maxwell® RSC 48
Kit: Promega Maxwell® Viral Total Nucleic Acid Purification Kit
Protocol: Viral Total Nucleic Acid
Recommendation(s): Add 120 µL of sample to 330 µL of pre-aliquoted External Lysis Buffer (300 µL
Lysis Buffer plus 30 µL Proteinase K; supplied within the kit) (total input volume is 450 µL). Proceed
with the extraction on the Maxwell® RSC 48. Elution volume is 75 µL.
Equivalence and performance of the following extraction platforms were demonstrated with the CDC
Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (K190302) and based on those data are
acceptable for use with the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel.
QIAGEN QIAcube
Kit: QIAGEN QIAamp® DSP Viral RNA Mini Kit or QIAamp® Viral RNA Mini Kit
Recommendations: Utilize 140 μL of sample and elute with 100 μL of buffer.
Roche MagNA Pure LC
Kit: Roche MagNA Pure Total Nucleic Acid Kit
Protocol: Total NA External_lysis
Recommendation(s): Add 100 μL of sample to 300 μL of pre-aliquoted TNA isolation kit lysis buffer
(total input sample volume is 400 μL). Elution volume is 100 μL.
Roche MagNA Pure Compact
Kit: Roche MagNA Pure Nucleic Acid Isolation Kit I
Protocol: Total_NA_Plasma100_400
Recommendation(s): Add 100 μL of sample to 300 μL of pre-aliquoted TNA isolation kit lysis buffer
(total input sample volume is 400 μL). Elution volume is 100 μL.
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bioMérieux NucliSENS® easyMAG® Instrument
Protocol: General protocol (not for blood) using “Off-board Lysis” reagent settings.
Recommendation(s): Add 100 μL of sample to 1000 μL of pre-aliquoted easyMAG lysis buffer (total
input sample volume is 1100 μL). Incubate for 10 minutes at room temperature. Elution volume is 100
μL.
bioMérieux EMAG® Instrument
Protocol: Custom protocol: CDC Flu V1 using “Off-board Lysis” reagent settings.
Recommendation(s): Add 100 μL of samples to 2000 μL of pre-aliquoted easyMAG lysis buffer (total
input sample volume is 2100 μL). Incubate for 10 minutes at room temperature. Elution volume is 100
μL. The custom protocol, CDC Flu V1, is programmed on the bioMérieux EMAG® instrument with the
assistance of a bioMérieux service representative. Installation verification is documented at the time of
installation. Laboratories are recommended to retain a record of the step-by-step verification of the
bioMérieux custom protocol installation procedure.
Manufacturer’s recommended procedures (except as noted in recommendations above) are to be
followed for sample extraction. HSC must be included in each extraction batch.
Disclaimer: Names of vendors or manufacturers are provided as examples of suitable product sources. Inclusion
does not imply endorsement by the Centers for Disease Control and Prevention.
Assay Set Up
Reaction Master Mix and Plate Set Up
Note: Plate set-up configuration can vary with the number of specimens and workday
organization. NTCs and nCoVPCs must be included in each run.
1) In the reagent set-up room clean hood, place rRT-PCR buffer, enzyme, and primer/probes on
ice or cold-block. Keep cold during preparation and use.
2) Mix buffer, enzyme, and primer/probes by inversion 5 times.
3) Centrifuge reagents and primers/probes for 5 seconds to collect contents at the bottom of
the tube, and then place the tube in a cold rack.
4) Label one 1.5 mL microcentrifuge tube for each primer/probe set.
5) Determine the number of reactions (N) to set up per assay. It is necessary to make excess
reaction mix for the NTC, nCoVPC, HSC (if included in the RT-PCR run), and RP reactions and
for pipetting error. Use the following guide to determine N:
• If number of samples (n) including controls equals 1 through 14, then N = n + 1
• If number of samples (n) including controls is 15 or greater, then N = n + 2
7) For each primer/probe set, calculate the amount of each reagent to be added for each
reaction mixture (N = # of reactions).
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Thermofisher TaqPath™ 1-Step RT-qPCR Master Mix
Step # Reagent Vol. of Reagent Added
per Reaction
1 Nuclease-free Water N x 8.5 µL
2 Combined Primer/Probe Mix N x 1.5 µL
3 TaqPathTM 1-Step RT-qPCR Master Mix (4x) N x 5.0 µL
Total Volume N x 15.0 µL
Promega GoTaq® Probe 1- Step RT-qPCR System
Step # Reagent Vol. of Reagent Added
per Reaction
1 Nuclease-free Water N x 3.1 µL
2 Combined Primer/Probe Mix N x 1.5 µL
3 GoTaq Probe qPCR Master Mix with dUTP N x 10.0 µL
4 Go Script RT Mix for 1-Step RT-qPCR N x 0.4 µL
Total Volume N x 15.0 µL
Quantabio qScript XLT One-Step RT-qPCR ToughMix
Step # Reagent Vol. of Reagent Added
per Reaction
1 Nuclease-free Water N x 3.5 µL
2 Combined Primer/Probe Mix N x 1.5 µL
3 qScript XLT One-Step RT-qPCR ToughMix
(2X)
N x 10.0 µL
Total Volume N x 15.0 µL
Quantabio UltraPlex 1-Step ToughMix (4X)
Step # Reagent Vol. of Reagent Added
per Reaction
1 Nuclease-free Water N x 8.5 µL
2 Combined Primer/Probe Mix N x 1.5 µL
3 UltraPlex 1-Step ToughMix (4X) N x 5.0 µL
Total Volume N x 15.0 µL
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8) Dispense reagents into each respective labeled 1.5 mL microcentrifuge tube. After addition of
the reagents, mix reaction mixtures by pipetting up and down. Do not vortex.
9) Centrifuge for 5 seconds to collect contents at the bottom of the tube, and then place the
tube in a cold rack.
10) Set up reaction strip tubes or plates in a 96-well cooler rack.
11) Dispense 15 µL of each master mix into the appropriate wells going across the row as shown
below (Figure 1):
Figure 1: Example of Reaction Master Mix Plate Set-Up
1 2 3 4 5 6 7 8 9 10 11 12
A N1 N1 N1 N1 N1 N1 N1 N1 N1 N1 N1 N1
B N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 N2
C RP RP RP RP RP RP RP RP RP RP RP RP
D
E
F
G
H
12) Prior to moving to the nucleic acid handling area, prepare the No Template Control (NTC)
reactions for column #1 in the assay preparation area.
13) Pipette 5 µL of nuclease-free water into the NTC sample wells (Figure 2, column 1). Securely
cap NTC wells before proceeding.
14) Cover the entire reaction plate and move the reaction plate to the specimen nucleic acid
handling area.
Nucleic Acid Template Addition
1) Gently vortex nucleic acid sample tubes for approximately 5 seconds.
2) Centrifuge for 5 seconds to collect contents at the bottom of the tube.
3) After centrifugation, place extracted nucleic acid sample tubes in the cold rack.
4) Samples should be added to columns 2-11 (column 1 and 12 are for controls) to the specific
assay that is being tested as illustrated in Figure 2. Carefully pipette 5.0 µL of the first sample
into all the wells labeled for that sample (i.e. Sample “S1” down column #2). Keep other
sample wells covered during addition. Change tips after each addition.
5) Securely cap the column to which the sample has been added to prevent cross contamination
and to ensure sample tracking.
6) Change gloves often and when necessary to avoid contamination.
7) Repeat steps #4 and #5 for the remaining samples.
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8) If necessary, add 5 µL of Human Specimen Control (HSC) extracted sample to the HSC wells
(Figure 2, column 11). Securely cap wells after addition. NOTE: Per CLIA regulations, HSC
must be tested at least once per day.
9) Cover the entire reaction plate and move the reaction plate to the positive template control
handling area.
Assay Control Addition
1) Pipette 5 µL of nCoVPC RNA to the sample wells of column 12 (Figure 2). Securely cap wells
after addition of the control RNA.
NOTE: If using 8-tube strips, label the TAB of each strip to indicate sample position. DO NOT
LABEL THE TOPS OF THE REACTION TUBES!
2) Briefly centrifuge reaction tube strips for 10-15 seconds. After centrifugation return to cold
rack.
NOTE: If using 96-well plates, centrifuge plates for 30 seconds at 500 x g, 4°C.
Figure 2. 2019-nCoV rRT-PCR Diagnostic Panel: Example of Sample and Control Set-up
1 2 3 4 5 6 7 8 9 10 11a 12
A NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 nCoV PC
B NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 nCoV PC
C NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 nCoV PC
D
E
F
G
H
a
Replace the sample in this column with extracted HSC if necessary
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Create a Run Template on the Applied Biosystems 7500 Fast Dx Real-time PCR
Instrument (Required if no template exists)
If the template already exists on your instrument, please proceed to the RUNNING A TEST section.
1) Launch the Applied Biosystems 7500 Fast Dx Real-time PCR Instrument by double clicking on the
Applied Biosystems 7500 Fast Dx System icon on the desktop.
2) A new window should appear, select Create New Document from the menu.
Figure 3. New Document Wizard Window
3) The New Document Wizard screen in Figure 3 will appear. Select:
a. Assay: Standard Curve (Absolute Quantitation)
b. Container: 96-Well Clear
c. Template: Blank Document
d. Run Mode: Standard 7500
e. Operator: Your Name
f. Comments: SDS v1.4
g. Plate Name: Your Choice
4) After making selections click Next at the bottom of the window.
Make sure to change
Run Mode to
STANDARD 7500
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Figure 4. Creating New Detectors
NOTE: ROX is the default passive reference. This will be changed to “none” in step 12.
5) After selecting next, the Select Detectors screen (Figure 4) will appear.
6) Click the New Detector button (see Figure 4).
7) The New Detector window will appear (Figure 5). A new detector will need to be defined for
each primer and probe set. Creating these detectors will enable you to analyze each primer and
probe set individually at the end of the reaction.
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Figure 5. New Detector Window
8) Start by creating the N1 Detector. Include the following:
a. Name: N1
b. Description: leave blank
c. Reporter Dye: FAM
d. Quencher Dye: (none)
e. Color: to change the color of the detector indicator do the following:
⇒ Click on the color square to reveal the color chart
⇒ Select a color by clicking on one of the squares
⇒ After selecting a color click OK to return to the New Detector screen
f. Click the OK button of the New Detector screen to return to the screen shown in Figure 4.
9) Repeat step 6-8 for each target in the panel.
Name Reporter Dye Quencher Dye
N1 FAM (none)
N2 FAM (none)
RP FAM (none)
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10) After each Detector is added, the Detector Name, Description, Reporter and Quencher fields
will become populated in the Select Detectors screen (Figure 6).
11) Before proceeding, the newly created detectors must be added to the document. To add the
new detectors to the document, click ADD (see Figure 6). Detector names will appear on the
right-hand side of the Select Detectors window (Figure 6).
Figure 6. Adding New Detectors to Document
12) Once all detectors have been added, select (none) for Passive Reference at the top right-hand
drop-down menu (Figure 7).
Figure 7. Select Passive Reference
Passive reference should be set to “(none)” as described above.
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13)Click Next at the bottom of the Select Detectors window to proceed to the Set Up Sample Plate
window (Figure 8).
14)In the Set Up Sample Plate window (Figure 8), use your mouse to select row A from the lower
portion of the window, in the spreadsheet (see Figure 8).
15)In the top portion of the window, select detector N1. A check will appear next to the detector
you have selected (Figure 8). You will also notice the row in the spreadsheet will be populated
with a colored “U” icon to indicate which detector you’ve selected.
16)Repeat step 14-15 for each detector that will be used in the assay.
Figure 8. Sample Plate Set-up
17) Select Finish after detectors have been assigned to their respective rows. (Figure 9).
Figure 9. Finished Plate Set-up
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18) After clicking “Finish”, there will be a brief pause allowing the Applied Biosystems 7500 Fast Dx
to initialize. This initialization is followed by a clicking noise. Note: The machine must be turned
on for initialization.
19) After initialization, the Plate tab of the Setup (Figure 10) will appear.
20)Each well of the plate should contain colored U icons that correspond with the detector labels
that were previously chosen. To confirm detector assignments, select Tools from the file menu,
then select Detector Manager.
Figure 10. Plate Set-up Window
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21) The Detector Manager window will appear (Figure 11).
Figure 11. Detector Manager Window
22)Confirm all detectors are included and that each target has a Reporter set to FAM and the
Quencher is set to (none).
23)If all detectors are present, select Done. The detector information has been created and
assigned to wells on the plate.
Defining the Instrument Settings
1) After detectors have been created and assigned, proceed to instrument set up.
2) Select the Instrument tab to define thermal cycling conditions.
3) Modify the thermal cycling conditions as follows (Figure 12):
Thermofisher TaqPath™ 1-Step RT-qPCR Master Mix, CG
a. In Stage 1, Set to 2 min at 25°C; 1 Rep.
b. In Stage 2, Set to 15 min at 50°C; 1 Rep.
c. In Stage 3, Set to 2 min at 95°C, 1 Rep.
d. In Stage 4, Step 1 set to 3 sec at 95°C.
e. In Stage 4, Step 2 set to 30 sec at 55.0°C.
f. In Stage 4, Reps should be set to 45.
g. Under Settings (Figure 12), bottom left-hand box, change volume to 20 µL.
h. Under Settings, Run Mode selection should be Standard 7500.
i. Step 2 of Stage 4 should be highlighted in yellow to indicate data collection (see Figure
12).
OR
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Quantabio qScriptTM XLT One-Step RT-qPCR ToughMix or UltraPlex 1-Step ToughMix (4X)
a. In Stage 1, Set to 10 min at 50°C; 1 Rep.
b. In Stage 2, Set to 3 min at 95°C, 1 Rep.
c. In Stage 3, Step 1 set to 3 sec at 95°C.
d. In Stage 3, Step 2 set to 30 sec at 55.0°C.
e. In Stage 3, Reps should be set to 45.
f. Under Settings (Figure 12), bottom left-hand box, change volume to 20 µL.
g. Under Settings, Run Mode selection should be Standard 7500.
h. Step 2 of Stage 3 should be highlighted in yellow to indicate data collection (see Figure
12).
OR
Promega GoTaq® Probe 1-Step RT-qPCR System
a. In Stage 1, Set to 15 min at 45°C; 1 Rep.
b. In Stage 2, Set to 2 min at 95°C, 1 Rep.
c. In Stage 3, Step 1 set to 3 sec at 95°C.
d. In Stage 3, Step 2 set to 30 sec at 55.0°C.
e. In Stage 3, Reps should be set to 45.
f. Under Settings (Figure 12), bottom left-hand box, change volume to 20 µL.
g. Under Settings, Run Mode selection should be Standard 7500.
h. Step 2 of Stage 3 should be highlighted in yellow to indicate data collection (see Figure
12).
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Figure 12. Instrument Window
4) After making changes to the Instrument tab, the template file is ready to be saved. To save the template,
select File from the top menu, then select Save As. Since the enzyme options have different instrument
settings, it is recommended that the template be saved with a name indicating the enzyme option.
5) Save the template as 2019-nCoV Dx Panel TaqPath or 2019-nCoV Dx Panel Quanta or 2019-nCoV Dx
Panel Promega as appropriate in the desktop folder labeled “ABI Run Templates” (you must create this
folder). Save as type should be SDS Templates (*.sdt) (Figure 13).
Figure 13. Saving Template
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Running a Test
1) Turn on the ABI 7500 Fast Dx Real-Time PCR Instrument.
2) Launch the Applied Biosystems 7500 Fast Dx Real-time PCR System by double clicking on the
7500 Fast Dx System icon on the desktop.
3) A new window should appear, select Open Existing Document from the menu.
4) Navigate to select your ABI Run Template folder from the desktop.
5) Double click on the appropriate template file (2019-nCoV Dx Panel TaqPath or 2019-nCoV Dx
Panel Quanta or 2019-nCoV Dx Panel Promega)
6) There will be a brief pause allowing the Applied Biosystems 7500 Fast Dx Real-Time PCR
Instrument to initialize. This initialization is followed by a clicking noise. Note: The machine must
be turned on for initialization.
Figure 14. Plate Set-up Window
7) After the instrument initializes, a plate map will appear (Figure 14). The detectors and controls
should already be labeled as they were assigned in the original template.
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8) Click the Well Inspector icon from the top menu.
9) Highlight specimen wells of interest on the plate map.
10) Type sample identifiers to Sample Name box in the Well Inspector window (Figure 15).
Figure 15. Labeling Wells
11) Repeat steps 9-10 until all sample identifiers are added to the plate setup.
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12) Once all specimen and control identifiers are added click the Close button on the Well Inspector
window to return to the Plate set up tab.
13) Click the Instrument tab at the upper left corner.
14) The reaction conditions, volumes, and type of 7500 reaction should already be loaded (Figure
16).
Figure 16. Instrument Settings
15) Ensure settings are correct (refer to the Defining Instrument Settings).
16) Before proceeding, the run file must be saved; from the main menu, select File, then Save As.
Save in appropriate run folder designation.
17) Load the plate into the plate holder in the instrument. Ensure that the plate is properly aligned
in the holder.
18) Once the run file is saved, click the Start button. Note: The run should take approximately 1 hour
and 20 minutes to complete.
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Data Analysis
1) After the run has completed, select the Results tab at the upper left corner of the software.
2) Select the Amplification Plot tab to view the raw data (Figure 17).
Figure 17. Amplification Plot Window
3) Start by highlighting all the samples from the run; to do this, click on the upper left-hand box (a)
of the sample wells (Figure 17). All the growth curves should appear on the graph.
4) On the right-hand side of the window (b), the Data drop down selection should be set to Delta
Rn vs. Cycle.
5) Select N1 from (c), the Detector drop down menu, using the downward arrow.
a. Please note that each detector is analyzed individually to reflect different
performance profiles of each primer and probe set.
6) In the Line Color drop down (d), Detector Color should be selected.
7) Under Analysis Settings select Manual Ct (e).
b. Do not change the Manual Baseline default numbers.
8) Using the mouse, click and drag the red threshold line until it lies within the exponential phase
of the fluorescence curves and above any background signal (Figure 18).
c
c
a
b
c
d
e
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Figure 18. Amplification Plot
9) Click the Analyze button in the lower right corner of the window. The red threshold line will turn
to green, indicating the data has been analyzed.
10) Repeat steps 5-9 to analyze results generated for each set of markers (N1, N2, RP).
11) Save analysis file by selecting File then Save As from the main menu.
12) After completing analysis for each of the markers, select the Report tab above the graph to
display the Ct values (Figure 19). To filter report by sample name in ascending or descending
order, simply click on Sample Name in the table.
Figure 19. Report
Exponential
PCR Phase
Background noise
Threshold adjusted
to fall within the
PCR exponential
phase.
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Interpretation of Results and Reporting
Extraction and Positive Control Results and Interpretation
No Template Control (NTC)
The NTC consists of using nuclease-free water in the rRT-PCR reactions instead of RNA. The NTC
reactions for all primer and probe sets should not exhibit fluorescence growth curves that cross the
threshold line. If any of the NTC reactions exhibit a growth curve that crosses the cycle threshold, sample
contamination may have occurred. Invalidate the run and repeat the assay with strict adherence to the
guidelines.
2019-nCoV Positive Control (nCoVPC)
The nCoVPC consists of in vitro transcribed RNA. The nCoVPC will yield a positive result with the
following primer and probe sets: N1, N2, and RP.
Human Specimen Control (HSC) (Extraction Control)
When HSC is run with the CDC 2019-nCoV rRT-PCR Diagnostic Panel (see previous section on Assay Set
Up), the HSC is used as an nucleic acid extraction procedural control to demonstrate successful recovery
of nucleic acid as well as extraction reagent integrity. The HSC control consists of noninfectious cultured
human cell (A549) material. Purified nucleic acid from the HSC should yield a positive result with the RP
primer and probe set and negative results with all 2019-nCoV markers.
Expected Performance of Controls Included in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Control
Type
External
Control
Name
Used to
Monitor
2019
nCoV_N1
2019
nCoV_N2 RP Expected Ct
Values
Positive nCoVPC
Substantial
reagent failure
including
primer and
probe integrity
+ + + < 40.00 Ct
Negative NTC
Reagent and/or
environmental
contamination
- - - None
detected
Extraction HSC
Failure in lysis
and extraction
procedure,
potential
contamination
during
extraction
- - + < 40.00 Ct
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If any of the above controls do not exhibit the expected performance as described, the assay may have
been set up and/or executed improperly, or reagent or equipment malfunction could have occurred.
Invalidate the run and re-test.
RNase P (Extraction Control)
 All clinical samples should exhibit fluorescence growth curves in the RNase P reaction that cross
the threshold line within 40.00 cycles (< 40.00 Ct), thus indicating the presence of the human
RNase P gene. Failure to detect RNase P in any clinical specimens may indicate:
− Improper extraction of nucleic acid from clinical materials resulting in loss of RNA and/or
RNA degradation.
− Absence of sufficient human cellular material due to poor collection or loss of specimen
integrity.
− Improper assay set up and execution.
− Reagent or equipment malfunction.
 If the RP assay does not produce a positive result for human clinical specimens, interpret as
follows:
− If the 2019-nCoV N1 and N2are positive even in the absence of a positive RP, the result
should be considered valid. It is possible, that some samples may fail to exhibit RNase P
growth curves due to low cell numbers in the original clinical sample. A negative RP signal
does not preclude the presence of 2019-nCoV virus RNA in a clinical specimen.
− If all 2019-nCoV markers AND RNase P are negative for the specimen, the result should be
considered invalid for the specimen. If residual specimen is available, repeat the extraction
procedure and repeat the test. If all markers remain negative after re-test, report the
results as invalid and a new specimen should be collected if possible.
2019-nCoV Markers (N1 and N2)
• When all controls exhibit the expected performance, a specimen is considered negative if all
2019-nCoV marker (N1, N2) cycle threshold growth curves DO NOT cross the threshold line
within 40.00 cycles (< 40.00 Ct) AND the RNase P growth curve DOES cross the threshold line
within 40.00 cycles (< 40.00 Ct).
• When all controls exhibit the expected performance, a specimen is considered positive for 2019-
nCoV if all 2019-nCoV marker (N1, N2) cycle threshold growth curves cross the threshold line
within 40.00 cycles (< 40.00 Ct). The RNase P may or may not be positive as described above, but
the 2019-nCoV result is still valid.
• When all controls exhibit the expected performance and the growth curves for the 2019-nCoV
markers (N1, N2) AND the RNase P marker DO NOT cross the cycle threshold growth curve within
40.00 cycles (< 40.00 Ct), the result is invalid. The extracted RNA from the specimen should be retested. If residual RNA is not available, re-extract RNA from residual specimen and re-test. If the
re-tested sample is negative for all markers and RNase P, the result is invalid and collection of a
new specimen from the patient should be considered.
• When all controls exhibit the expected performance and the cycle threshold growth curve for any
one marker (N1 or N2, but not both markers) crosses the threshold line within 40.00 cycles (<
40.00 Ct) the result is inconclusive. The extracted RNA should be retested. If residual RNA is not
available, re-extract RNA from residual specimen and re-test. If the same result is obtained,
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report the inconclusive result. Consult with your state public health laboratory or CDC, as
appropriate, to request guidance and/or to coordinate transfer of the specimen for additional
analysis.
• If HSC is positive for N1 or N2, then contamination may have occurred during extraction or
sample processing. Invalidate all results for specimens extracted alongside the HSC. Re-extract
specimens and HSC and re-test.
2019-nCoV rRT-PCR Diagnostic Panel Results Interpretation Guide
The table below lists the expected results for the 2019-nCoV rRT-PCR Diagnostic Panel. If a laboratory
obtains unexpected results for assay controls or if inconclusive or invalid results are obtained and cannot
be resolved through the recommended re-testing, please contact CDC for consultation and possible
specimen referral. See pages 13 and 50 for referral and contact information.
2019
nCoV_N1
2019
nCoV_N2 RP Result
Interpretationa Report Actions
+ + ±
2019-nCoV
detected Positive 2019-nCoV
Report results to CDC and
sender.
If only one of the two
targets is positive ±
Inconclusive
Result Inconclusive
Repeat testing of nucleic acid
and/or re-extract and repeat
rRT-PCR. If the repeated result
remains inconclusive, contact
your State Public Health
Laboratory or CDC for
instructions for transfer of the
specimen or further guidance.
- - +
2019-nCoV not
detected Not Detected
Report results to sender.
Consider testing for other
respiratory viruses.b
- - - Invalid Result Invalid
Repeat extraction and rRT-PCR.
If the repeated result remains
invalid, consider collecting a
new specimen from the patient. aLaboratories should report their diagnostic result as appropriate and in compliance with their specific reporting
system.
bOptimum specimen types and timing for peak viral levels during infections caused by 2019-nCoV have not been
determined. Collection of multiple specimens from the same patient may be necessary to detect the virus. The
possibility of a false negative result should especially be considered if the patient’s recent exposures or clinical
presentation suggest that 2019-nCoV infection is possible, and diagnostic tests for other causes of illness (e.g.,
other respiratory illness) are negative. If 2019-nCoV infection is still suspected, re-testing should be considered
in consultation with public health authorities.
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Quality Control
• Quality control requirements must be performed in conformance with local, state, and federal
regulations or accreditation requirements and the user’s laboratory’s standard quality control
procedures. For further guidance on appropriate quality control practices, refer to 42 CFR
493.1256.
• Quality control procedures are intended to monitor reagent and assay performance.
• Test all positive controls prior to running diagnostic samples with each new kit lot to ensure all
reagents and kit components are working properly.
• Good laboratory practice (cGLP) recommends including a positive extraction control in each
nucleic acid isolation batch.
• Although HSC is not included with the 2019-nCov rRT-PCR Diagnostic Panel, the HSC extraction
control must proceed through nucleic acid isolation per batch of specimens to be tested.
• Always include a negative template control (NTC) and the appropriate positive control (nCoVPC)
in each amplification and detection run. All clinical samples should be tested for human RNase P
gene to control for specimen quality and extraction.
Limitations
• All users, analysts, and any person reporting diagnostic results should be trained to perform this
procedure by a competent instructor. They should demonstrate their ability to perform the test
and interpret the results prior to performing the assay independently.
• Performance of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel has only been established
in upper and lower respiratory specimens (such as nasopharyngeal or oropharyngeal swabs,
sputum, lower respiratory tract aspirates, bronchoalveolar lavage, and nasopharyngeal
wash/aspirate or nasal aspirate).
• Negative results do not preclude 2019-nCoV infection and should not be used as the sole basis
for treatment or other patient management decisions. Optimum specimen types and timing for
peak viral levels during infections caused by 2019-nCoV have not been determined. Collection of
multiple specimens (types and time points) from the same patient may be necessary to detect
the virus.
• A false-negative result may occur if a specimen is improperly collected, transported or handled.
False-negative results may also occur if amplification inhibitors are present in the specimen or if
inadequate numbers of organisms are present in the specimen.
• Positive and negative predictive values are highly dependent on prevalence. False-negative test
results are more likely when prevalence of disease is high. False-positive test results are more
likely when prevalence is moderate to low.
• Do not use any reagent past the expiration date.
• If the virus mutates in the rRT-PCR target region, 2019-nCoV may not be detected or may be
detected less predictably. Inhibitors or other types of interference may produce a false-negative
result. An interference study evaluating the effect of common cold medications was not
performed.
• Test performance can be affected because the epidemiology and clinical spectrum of infection
caused by 2019-nCoV is not fully known. For example, clinicians and laboratories may not know
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the optimum types of specimens to collect, and, during the course of infection, when these
specimens are most likely to contain levels of viral RNA that can be readily detected.
• Detection of viral RNA may not indicate the presence of infectious virus or that 2019-nCoV is the
causative agent for clinical symptoms.
• The performance of this test has not been established for monitoring treatment of 2019-nCoV
infection.
• The performance of this test has not been established for screening of blood or blood products
for the presence of 2019-nCoV.
• This test cannot rule out diseases caused by other bacterial or viral pathogens.
Conditions of Authorization for the Laboratory
The CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel Letter of Authorization, along with the
authorized Fact Sheet for Healthcare Providers, the authorized Fact Sheet for Patients, and authorized
labeling are available on the FDA website:
https://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm
Use of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel must follow the procedures outlined in
these manufacturer’s Instructions for Use and the conditions of authorization outlined in the Letter of
Authorization. Deviations from the procedures outlined are not permitted under the Emergency Use
Authorization (EUA). To assist clinical laboratories running the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel, the relevant Conditions of Authorization are listed verbatim below, and are required to
be met by laboratories performing the EUA test.
• Authorized laboratories1 will include with reports of the results of the CDC 2019-nCoV Real-Time
RT-PCR Diagnostic Panel, all authorized Fact Sheets. Under exigent circumstances, other
appropriate methods for disseminating these Fact Sheets may be used, which may include mass
media.
• Authorized laboratories will perform the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel as
outlined in the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel
Instructions for Use. Deviations from the authorized procedures, including the authorized RT-PCR
instruments, authorized extraction methods, authorized clinical specimen types, authorized
control materials, authorized other ancillary reagents and authorized materials required to
perform the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel are not permitted. 2
• Authorized laboratories that receive the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel must
notify the relevant public health authorities of their intent to run the test prior to initiating
testing.
1Authorized Laboratories: For ease of reference, the Letter of Authorization refers to “laboratories certified under the Clinical
Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. § 263a, to perform high complexity tests” as “authorized
laboratories.”
2If an authorized laboratory is interested in implementing changes to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
that are not in the scope (Section II) of this letter of authorization FDA recommends you discuss with FDA after considering
the policy outlined in Immediately in Effect Guidance for Clinical Laboratories and Food and Drug Administration Staff: Policy
for Diagnostics Testing in Laboratories Certified to Perform High Complexity Testing under CLIA prior to Emergency Use
Authorization for Coronavirus Disease-2019 during the Public Health Emergency
(https://www.fda.gov/media/135659/download).
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• Authorized laboratories will have a process in place for reporting test results to healthcare
providers and relevant public health authorities, as appropriate.
• Authorized laboratories will collect information on the performance of the test and report to
DMD/OHT7-OIR/OPEQ/CDRH (via email: CDRH-EUA-Reporting@fda.hhs.gov) and CDC
(respvirus@cdc.gov) any suspected occurrence of false-positive or false-negative results and
significant deviations from the established performance characteristics of the test of which they
become aware.
• Authorized laboratories will report adverse events, including problems with test performance or
results, to MedWatch by submitting the online FDA Form 3500
(https://www.accessdata.fda.gov/scripts/medwatch/index.cfm?action=reporting.home) or by
calling 1-800-FDA-1088
• All laboratory personnel using the test must be appropriately trained in RT-PCR techniques and
use appropriate laboratory and personal protective equipment when handling this kit and use
the test in accordance with the authorized labeling.
• CDC, IRR, manufacturers and distributors of commercial materials identified as acceptable on the
CDC website, and authorized laboratories will ensure that any records associated with this EUA
are maintained until otherwise notified by FDA. Such records will be made available to FDA for
inspection upon request.
Performance Characteristics
Analytical Performance:
Limit of Detection (LoD):
LoD studies determine the lowest detectable concentration of 2019-nCoV at which approximately 95%
of all (true positive) replicates test positive. The LoD was determined by limiting dilution studies using
characterized samples.
The analytical sensitivity of the rRT-PCR assays contained in the CDC 2019 Novel Coronavirus (2019-
nCoV) Real-Time RT-PCR Diagnostic Panel were determined in Limit of Detection studies. Since no
quantified virus isolates of the 2019-nCoV are currently available, assays designed for detection of the
2019-nCoV RNA were tested with characterized stocks of in vitro transcribed full length RNA (N gene;
GenBank accession: MN908947.2) of known titer (RNA copies/µL) spiked into a diluent consisting of a
suspension of human A549 cells and viral transport medium (VTM) to mimic clinical specimen. Samples
were extracted using the QIAGEN EZ1 Advanced XL instrument and EZ1 DSP Virus Kit (Cat# 62724) and
manually with the QIAGEN DSP Viral RNA Mini Kit (Cat# 61904). Real-Time RT-PCR assays were
performed using the ThemoFisher Scientific TaqPath™ 1-Step RT-qPCR Master Mix, CG (Cat# A15299) on
the Applied Biosystems™ 7500 Fast Dx Real-Time PCR Instrument according to the CDC 2019-nCoV RealTime RT-PCR Diagnostic Panel instructions for use.
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A preliminary LoD for each assay was determined testing triplicate samples of RNA purified using each
extraction method. The approximate LoD was identified by extracting and testing 10-fold serial dilutions
of characterized stocks of in vitro transcribed full-length RNA. A confirmation of the LoD was determined
using 3-fold serial dilution RNA samples with 20 extracted replicates. The LoD was determined as the
lowest concentration where ≥ 95% (19/20) of the replicates were positive.
Table 4. Limit of Detection Confirmation of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
with QIAGEN EZ1 DSP
Targets 2019-nCoV_N1 2019-nCoV_N2
RNA Concentration1 10 0.5 10 0.0 10 -0.5 10 0.5 10 0.0 10 -0.5
Positives/Total 20/20 19/20 13/20 20/20 17/20 9/20
Mean Ct2 32.5 35.4 NA 35.8 NA NA
Standard Deviation
(Ct) 0.5 0.8 NA 1.3 NA NA
1 Concentration is presented in RNA copies/µL
2 Mean Ct reported for dilutions that are ≥ 95% positive. Calculations only include positive results.
NA not applicable
Table 5. Limit of Detection Confirmation CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel with
QIAGEN QIAmp DSP Viral RNA Mini Kit
Targets 2019-nCoV_N1 2019-nCoV_N2
RNA Concentration1 10 0.5 10 0.0 10 -0.5 10 0.5 10 0.0 10 -0.5 10 -1.0
Positives/Total 20/20 20/20 6/20 20/20 20/20 20/20 8/20
Mean Ct2 32.0 32.8 NA 33.0 35.4 36.2 NA
Standard Deviation
(Ct) 0.7 0.8 NA 1.4 0.9 1.9 NA
1 Concentration is presented in RNA copies/µL
2 Mean Ct reported for dilutions that are ≥ 95% positive. Calculations only include positive results.
NA not applicable
Table 6. Limit of Detection of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Virus Material
Limit of Detection (RNA copies/µL)
QIAGEN EZ1
Advanced XL
QIAGEN DSP Viral
RNA Mini Kit
2019 Novel
Coronavirus
N Gene RNA
Transcript 100.5 100
FDA Sensitivity Evaluation: The analytical sensitivity of the test will be further assessed by evaluating an
FDA-recommended reference material using an FDA developed protocol if applicable and/or when
available.
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In Silico Analysis of Primer and Probe Sequences:
The oligonucleotide primer and probe sequences of the CDC 2019 nCoV Real-Time RT-PCR Diagnostic
Panel were evaluated against 31,623 sequences available in the Global Initiative on Sharing All Influenza
Data (GISAID, https://www.gisaid.org) database as of June 20, 2020, to demonstrate the predicted
inclusivity of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. Nucleotide mismatches in the
primer/probe regions with frequencies > 0.1% are shown below. With the exception of one nucleotide
mismatch with frequency > 1% (2.00%) at the third position of the N1 probe, the frequency of all
mismatches was < 1%, indicating that prevalence of the mismatches were sporadic. Only one sequence
(0.0032%) had two nucleotide mismatches in the N1 probe, and one other sequence from a different
isolate (0.0032%) had two nucleotide mismatches in the N1 reverse primer. No sequences were found to
have more than one mismatch in any N2 primer/probe region. The risk of these mismatches resulting in
a significant loss in reactivity causing a false negative result is extremely low due to the design of the
primers and probes, with melting temperatures > 60°C and with annealing temperature at 55°C that can
tolerate up to two mismatches.
Table 7. In Silico Inclusivity Analysis of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel Among
31,623 Genome Sequences Available from GISAID as of June 20, 2020
Primer/probe N1 probe N1 reverse N2 probe
Location (5'>3') 3 15 21 13
Mismatch Nucleotide C>T G>T T>C C>T
Mismatch No. 632 34 71 46
Mismatch Frequency (%) 2.00 0.11 0.22 0.15
Specificity/Exclusivity Testing: In Silico Analysis
BLASTn analysis queries of the 2019-nCoV rRT-PCR assays primers and probes were performed against
public domain nucleotide sequences. The database search parameters were as follows: 1) The nucleotide
collection consists of GenBank+EMBL+DDBJ+PDB+RefSeq sequences, but excludes EST, STS, GSS, WGS,
TSA, patent sequences as well as phase 0, 1, and 2 HTGS sequences and sequences longer than 100Mb;
2) The database is non-redundant. Identical sequences have been merged into one entry, while
preserving the accession, GI, title and taxonomy information for each entry; 3) Database was updated on
10/03/2019; 4) The search parameters automatically adjust for short input sequences and the expect
threshold is 1000; 5) The match and mismatch scores are 1 and -3, respectively; 6) The penalty to create
and extend a gap in an alignment is 5 and 2 respectively.
2019-nCoV_N1 Assay:
Probe sequence of 2019-nCoV rRT-PCR assay N1 showed high sequence homology with SARS
coronavirus and Bat SARS-like coronavirus genome. However, forward and reverse primers showed no
sequence homology with SARS coronavirus and Bat SARS-like coronavirus genome. Combining primers
and probe, there is no significant homologies with human genome, other coronaviruses or human
microflora that would predict potential false positive rRT-PCR results.
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2019-nCoV_N2 Assay:
The forward primer sequence of 2019-nCoV rRT-PCR assay N2 showed high sequence homology to Bat
SARS-like coronaviruses. The reverse primer and probe sequences showed no significant homology with
human genome, other coronaviruses or human microflora. Combining primers and probe, there is no
prediction of potential false positive rRT-PCR results.
In summary, the 2019-nCoV rRT-PCR assay N1 and N2, designed for the specific detection of 2019-nCoV,
showed no significant combined homologies with human genome, other coronaviruses, or human
microflora that would predict potential false positive rRT-PCR results.
In addition to the in silico analysis, several organisms were extracted and tested with the CDC 2019-nCoV
Real-Time RT-PCR Diagnostic Panel to demonstrate analytical specificity and exclusivity. Studies were
performed with nucleic acids extracted using the QIAGEN EZ1 Advanced XL instrument and EZ1 DSP
Virus Kit. Nucleic acids were extracted from high titer preparations (typically ≥ 105 PFU/mL or ≥ 106
CFU/mL). Testing was performed using the ThemoFisher Scientific TaqPath™ 1-Step RT-qPCR Master Mix,
CG on the Applied Biosystems™ 7500 Fast Dx Real-Time PCR instrument. The data demonstrate that the
expected results are obtained for each organism when tested with the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel.
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Table 8. Specificity/Exclusivity of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Virus Strain Source
2019-
nCoV_
N1
2019-
nCoV_
N2
Final
Result
Human coronavirus 229E Isolate 0/3 0/3 Neg.
Human coronavirus OC43 Isolate 0/3 0/3 Neg.
Human coronavirus NL63 clinical specimen 0/3 0/3 Neg.
Human coronavirus HKU1 clinical specimen 0/3 0/3 Neg.
MERS-coronavirus Isolate 0/3 0/3 Neg.
SARS-coronavirus Isolate 0/3 0/3 Neg.
bocavirus - clinical specimen 0/3 0/3 Neg.
Mycoplasma pneumoniae Isolate 0/3 0/3 Neg.
Streptococcus Isolate 0/3 0/3 Neg.
Influenza A(H1N1) Isolate 0/3 0/3 Neg.
Influenza A(H3N2) Isolate 0/3 0/3 Neg.
Influenza B Isolate 0/3 0/3 Neg.
Human adenovirus, type 1 Ad71 Isolate 0/3 0/3 Neg.
Human metapneumovirus - Isolate 0/3 0/3 Neg.
respiratory syncytial virus Long A Isolate 0/3 0/3 Neg.
rhinovirus Isolate 0/3 0/3 Neg.
parainfluenza 1 C35 Isolate 0/3 0/3 Neg.
parainfluenza 2 Greer Isolate 0/3 0/3 Neg.
parainfluenza 3 C-43 Isolate 0/3 0/3 Neg.
parainfluenza 4 M-25 Isolate 0/3 0/3 Neg.
Endogenous Interference Substances Studies:
The CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel uses conventional well-established nucleic acid
extraction methods and based on our experience with CDC’s other EUA assays, including the CDC Novel
Coronavirus 2012 Real-time RT-PCR Assay for the presumptive detection of Middle East Respiratory
Syndrome Coronavirus (MERS-CoV) and the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic
Panel-Influenza A/H7 (Eurasian Lineage) Assay for the presumptive detection of novel influenza A (H7N9)
virus that are both intended for use with a number of respiratory specimens, we do not anticipate
interference from common endogenous substances.
Specimen Stability and Fresh-frozen Testing:
To increase the likelihood of detecting infection, CDC recommends collection of lower respiratory and
upper respiratory specimens for testing. If possible, additional specimen types (e.g., stool, urine) should
be collected and should be stored initially until decision is made by CDC whether additional specimen
sources should be tested. Specimens should be collected as soon as possible once a PUI is identified
regardless of symptom onset. Maintain proper infection control when collecting specimens. Store
specimens at 2-8°C and ship overnight to CDC on ice pack. Label each specimen container with the
patient’s ID number (e.g., medical record number), unique specimen ID (e.g., laboratory requisition
number), specimen type (e.g., nasal swabs) and the date the sample was collected. Complete a CDC
Form 50.34 for each specimen submitted.
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Clinical Performance:
As of February 22, 2020, CDC has tested 2071 respiratory specimens from persons under investigation
(PUI) in the U.S. using the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. Specimen types include
bronchial fluid/wash, buccal swab, nasal wash/aspirate, nasopharyngeal swab, nasopharyngeal/throat
swab, oral swab, sputum, oropharyngeal (throat) swab, swab (unspecified), and throat swab.
Table 9: Summary of CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel Data
Generated by Testing Human Respiratory Specimens Collected from PUI Subjects in the U.S.
Specimen Type
2019 nCoV
Negative
2019 nCoV
Positive Inconclusive Invalid Total
Bronchial
fluid/wash 2 0 0 0 2
Buccal swab 5 1 0 0 6
Nasal
wash/aspirate 6 0 0 0 6
Nasopharyngeal
swab 927 23 0 0 950
Nasopharyngeal
swab/throat
swab 4 0 0 0 4
Oral swab 476 9 0 0 485
Pharyngeal
(throat) swab 363 10 0 1 374
Sputum 165 5 0 0 170
Swab
(unspecified)1 71 1 0 0 72
Tissue (lung) 2 0 0 0 2
Total 2021 49 0 1 2071
1Actual swab type information was missing from these upper respiratory tract specimens.
Two thousand twenty-one (2021) respiratory specimens of the 2071 respiratory specimens tested
negative by the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. Forty-nine (49) of the 2071
respiratory specimens tested positive by the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. Only
one specimen (oropharyngeal (throat) swab) was invalid. Of the 49 respiratory specimens that tested
positive by the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel, seventeen (17) were confirmed by
genetic sequencing and/or virus culture (positive percent agreement = 17/17, 95% CI: 81.6%-100%)
During the early phase of the testing, a total of 117 respiratory specimens collected from 46 PUI subjects
were also tested with two analytically validated real-time RT-PCR assays that target separate and
independent regions of the nucleocapsid protein gene of the 2019-nCoV, N4 and N5 assays. The
nucleocapsid protein gene targets for the N4 and N5 assays are different and independent from the
nucleocapsid protein gene targets for the two RT-PCR assays included in the CDC 2019-nCoV Real-Time
RT-PCR Diagnostic Panel, N1 and N2. Any positive result from the N4 and/or the N5 assay was further
investigated by genetic sequencing.
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Performance of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel testing these 117 respiratory
specimens was estimated against a composite comparator. A specimen was considered comparator
negative if both the N4 and the N5 assays were negative. A specimen was considered comparator
positive when the N4 and/or the N5 assay generated a positive result, and the comparator positive
result(s) were further investigated and confirmed to be 2019-nCoV RNA positive by genetic sequencing.
Table 10: Percent Agreement of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel with the
Composite Comparator
CDC 2019-nCoV
Panel Result
Composite Comparator Result
Positive Negative
Positive 131 0
Inconclusive 0 0
Negative 0 104
1Composite comparator results were available for 13 of 49 CDC 2019-nCoV Panel positive specimens
only.
Positive percent agreement = 13/13 = 100% (95% CI: 77.2% - 100%)
Negative percent agreement = 104/104 = 100% (95% CI: 96.4% - 100%)
Enzyme Master Mix Evaluation:
The limit of detection equivalence between the ThermoFisher TaqPath™ 1-Step RT-qPCR Master Mix and
the following enzyme master mixes was evaluated: Quantabio qScript XLT One-Step RT-qPCR ToughMix,
Quantabio UltraPlex 1-Step ToughMix (4X), and Promega GoTaq® Probe 1- Step RT-qPCR System. Serial
dilutions of 2019 novel coronavirus (SARS CoV-2) transcript were tested in triplicate with the CDC 2019-
nCoV Real-time RT-PCR Diagnostic Panel using all four enzyme master mixes. Both manufactured
versions of oligonucleotide probe, BHQ and ZEN, were used in the comparison. The lowest detectable
concentration of transcript at which all replicates tested positive using the Quantabio qScript XLT OneStep RT-qPCR ToughMix and Quantabio UltraPlex 1-Step ToughMix (4X) was similar to that observed for
the ThemoFisher TaqPath™ 1-Step RT-qPCR Master Mix. The lowest detectable concentration of
transcript when using the Promega GoTaq® Probe 1- Step RT-qPCR System was one dilution above that
observed for the other candidates when evaluated with the BHQ version of the CDC assays. The
candidate master mixes all performed equivalently or at one dilution below the ThemoFisher TaqPath™
1-Step RT-qPCR Master Mix when evaluated with the ZEN version of the CDC assays.
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Table 11: Limit of Detection Comparison for Enzyme Master Mixes – BHQ Probe Summary Results
Copy Number
ThemoFisher TaqPath™
1-Step RT-qPCR Master
Mix
Quantabio qScript XLT
One-Step RT-qPCR
ToughMix
Quantabio UltraPlex 1-
Step ToughMix (4X)
Promega GoTaq® Probe
1- Step RT-qPCR System
2019-
nCoV_N1
2019-
nCoV_N2
2019-
nCoV_N1
2019-
nCoV_N2
2019-
nCoV_N1
2019-
nCoV_N2
2019-
nCoV_N1
2019-
nCoV_N2
102 copies/µL 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3
101 copies/µL 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3
100 copies/µL 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3
10-1 copies µL 2/3 0/3 1/3 1/3 1/3 1/3 0/3 0/3
Table 12: Limit of Detection Comparison for Enzyme Master Mixes – ZEN Probe Summary Results
Copy Number
ThemoFisher TaqPath™
1-Step RT-qPCR Master
Mix
Quantabio qScript XLT
One-Step RT-qPCR
ToughMix
Quantabio UltraPlex 1-
Step ToughMix (4X)
Promega GoTaq® Probe
1- Step RT-qPCR System
2019-
nCoV_N1
2019-
nCoV_N2
2019-
nCoV_N1
2019-
nCoV_N2
2019-
nCoV_N1
2019-
nCoV_N2
2019-
nCoV_N1
2019-
nCoV_N2
102 copies/µL 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3
101 copies/µL 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3
100 copies/µL 3/3 2/3 3/3 3/3 3/3 2/3 3/3 3/3
10-1 copies µL 1/3 1/3 0/3 0/3 0/3 1/3 1/3 1/3
Retrospective positive (18) and negative (17) clinical respiratory specimens were extracted using the
QIAGEN EZ1 Advanced XL instrument and EZ1 DSP Virus Kit and were tested with the CDC 2019-nCoV
Real-time RT-PCR Diagnostic Panel using the Quantabio qScript XLT One-Step RT-qPCR ToughMix,
Quantabio UltraPlex 1-Step ToughMix (4X), and Promega GoTaq® Probe 1- Step RT-qPCR System master
mixes. All three enzyme master mixes performed equivalently, demonstrating 100% positive and 100%
negative agreement with expected results and a 95% confidence interval of 82.4%-100% and 81.6%-
100%, respectively.
Table 13: Clinical Comparison – Retrospective Study Summary Results
CDC 2019-nCoV
Real-time RTPCR Diagnostic
Panel Result
Quantabio qScript XLT
One-Step RT-qPCR
ToughMix
Quantabio UltraPlex 1-Step
ToughMix (4X)
Promega GoTaq® Probe 1-
Step RT-qPCR System
Positive Negative Positive Negative Positive Negative
Positive 18 0 18 0 18 0
Negative 0 17 0 17 0 17
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Roche MagNA Pure 24 and MagNA Pure 96 Extraction Platform Evaluation:
Performance of the 2019-CoV Real-time RT-PCR Diagnostic Panel using the Roche MagNA Pure 24 and
MagNA Pure 96 extraction platforms was compared to performance with an authorized extraction
method. Serial dilutions of quantified inactivated SARS-CoV-2 virus (USA-WA1/2020; 100 RNA copies/µL)
in lysis buffer were added to pooled negative upper respiratory tract specimen matrix. Five samples of
each dilution were extracted in parallel with the QIAGEN EZ1 Advanced XL (EZ1 DSP Virus Kit Cat# 62724)
and the Roche MagNA Pure 24 (MagNA Pure 24 Total NA Isolation Kit Cat# 07658036001) and Roche
MagNA Pure 96 (MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume Kit Cat# 06543588001)
extraction platforms and evaluated using the 2019-nCoV Real-Time RT-PCR Diagnostic Panel and
ThermoFisher TaqPath™ 1-Step RT-qPCR Master Mix. The observed LoD was defined as the lowest
concentration at which 100% (5 out of 5 total) of all replicates tested positive for both primer/probe sets
(N1 and N2) in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. The acceptance criteria for
equivalence were defined as demonstrating an observed LoD either at the same endpoint or within a 3-
fold dilution. The results showed that both the MagNA Pure 24 and MagNA Pure 96 extraction platforms
performed equivalently or within one 3-fold dilution of the LoD observed when using the QIAGEN EZ1
Advanced XL extraction platform.
Table 14. Limit of Detection Comparison between the QIAGEN EZ1 Advanced XL, Roche MagNA Pure
96, and Roche MagNA Pure 24 Extraction Platforms using the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel
Platform Parameter 2019-nCoV_N1 Assay 2019-nCoV_N2 Assay Observed
LoD1
QIAGEN EZ1
Advanced XL
RNA copies/µL 101.0 100.5 100.0 101.0 100.5 100.0
100.5 # pos./total 5/5 5/5 5/5 5/5 5/5 3/5
Mean Ct2 34.0 35.0 36.3 33.9 36.6 NA
Std. Deviation 0.2 0.8 0.2 0.4 0.9 NA
Roche MagNA Pure
96
RNA copies/µL 101.0 100.5 100.0 101.0 100.5 100.0
100.5 # pos./total 5/5 5/5 5/5 5/5 5/5 2/5
Mean Ct2 33.3 34.6 36.1 33.2 35.7 NA
Std. Deviation 0.5 0.5 0.3 0.3 0.4 NA
Roche MagNA Pure
24
RNA copies/µL 101.0 100.5 100.0 101.0 100.5 100.0
101.0 # pos./total 5/5 3/5 3/5 5/5 5/5 5/5
Mean Ct2 34.4 NA NA 35.2 36.9 36.2
Std. Deviation 0.6 NA NA 0.5 1.0 0.8
1
Concentration is presented in RNA copies/µL. The observed LoD is the lowest concentration where both assays showed 100%
positive detection.
2
Mean Ct reported for dilutions that show 100% positivity. Calculations only include positive results.
NA = not applicable
Previously characterized clinical remainder specimens (14 positive and 15 negative) were extracted using
both the Roche MagNA Pure 96 and MagNA Pure 24 extraction platforms and evaluated using the 2019-
nCoV Real-Time RT-PCR Diagnostic Panel and ThermoFisher TaqPath™ 1-Step RT-qPCR Master Mix.
Acceptance criteria for clinical equivalence was defined as demonstrating 100% concurrence with
qualitative results shown with the authorized comparator method (QIAGEN EZ1 Advanced XL). Results
from this study showed 100% concurrence with the comparator method for both the Roche MagNA Pure
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96 and Roche MagNA Pure 24 extraction platforms when used with the CDC 2019-nCoV Real-Time RTPCR Diagnostic panel.
Table 15. Clinical Comparison Results – Retrospective Study Results
Test Platform
Test
Platform
Result
QIAGEN EZ1
Advanced XL Result Positive % Agreement (CI)1 Negative % Agreement (CI)1
Positive Negative
Roche MagNA
Pure 96
Positive 14 0 100.0 (78.5 – 100.0) 100.0 (79.6 – 100.0) Negative 0 15
Roche MagNA
Pure 24
Positive 14 0 100.0 (78.5 – 100.0) 100.0 (79.6 – 100.0) Negative 0 15 1 CI = 95% confidence interval
Promega Maxwell® RSC 48 Extraction Platform Evaluation:
Performance of the 2019-CoV Real-time RT-PCR Diagnostic Panel using the Promega Maxwell® RSC 48
extraction platform was compared to performance with an authorized extraction method. Serial
dilutions of quantified inactivated SARS-CoV-2 virus (USA-WA1/2020; 100 RNA copies/µL) in VTM were
added to pooled negative upper respiratory tract specimen matrix. Five samples of each dilution were
extracted in parallel with the QIAGEN EZ1® Advanced XL (EZ1 DSP Virus Kit Cat# 62724) and the Promega
Maxwell® RSC 48 (Promega Maxwell® Viral Total Nucleic Acid Purification Kit Cat# AS1330) extraction
platforms and evaluated using the 2019-nCoV Real-Time RT-PCR Diagnostic Panel and ThermoFisher
TaqPath™ 1-Step RT-qPCR Master Mix. The observed LoD was defined as the lowest concentration at
which 100% (5 out of 5 total) of all replicates tested positive for both primer/probe sets (N1 and N2) in
the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. The acceptance criteria for equivalence were
defined as demonstrating an observed LoD either at the same endpoint or within a 3-fold dilution. The
results showed that the performance of the Maxwell® RSC 48 extraction platform performed
equivalently or within one 3-fold dilution of the LoD observed when using the QIAGEN EZ1® Advanced XL
extraction platform.
Table 16. Limit of Detection Comparison Between the QIAGEN EZ1® Advanced XL and Promega
Maxwell® RSC 48 Extraction Platforms Using the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Platform Parameter 2019-nCoV_N1 Assay 2019-nCoV_N2 Assay Observed
LoD1
QIAGEN EZ1®
Advanced XL
RNA copies/µL 100.5 100.0 10-0.5 100.5 100.0 10-0.5
10 # pos./total 5/5 5/5 0/5 5/5 5/5 3/5 0.0
Mean Ct2 32.27 33.80 NA 35.13 36.41 NA
Std. Deviation 0.81 0.40 NA 0.81 0.40 NA
Promega Maxwell®
RSC 48
RNA copies/µL 100.5 100.0 10-0.5 100.5 100.0 10-0.5
10 # pos./total 5/5 5/5 3/5 5/5 5/5 5/5 0.0
Mean Ct2 31.11 32.97 NA 31.89 33.95 35.17
Std. Deviation 0.24 0.34 NA 0.24 0.35 0.65
1
Concentration is presented in RNA copies/µL. The observed LoD is the lowest concentration where both assays showed 100%
positive detection.
2
Mean cycle threshold (Ct) reported for dilutions that show 100% positivity. Calculations only include positive results.
NA = not applicable
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CDC-006-00019, Revision: 05 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 07/13/2020
Previously characterized clinical remainder specimens (15 positive and 15 negative) were extracted using
the Promega Maxwell® RSC 48 extraction platform alongside the currently authorized QIAGEN EZ1®
Advanced XL extraction platform and evaluated using the 2019-nCoV Real-Time RT-PCR Diagnostic Panel
and ThermoFisher TaqPath™ 1-Step RT-qPCR Master Mix. Results from the Maxwell® RSC 48 were
compared with the QIAGEN EZ1® Advanced XL extraction performed in parallel showing 100% (15/15)
qualitative concurrence on positive samples and 93.3% (14/15) qualitative concurrence on negative
samples. This evaluation showed that two originally negative (QIAGEN QIAamp® DSP Viral RNA Mini Kit)
specimens (Specimens 16 and 24) yielded an inconclusive result after extraction using the QIAGEN EZ1®
Advanced XL. Repeat of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel resolved one of the two
specimens (Specimen 24, negative result). The second specimen (Specimen 16) remained inconclusive.
Both these specimens yielded a negative result on the Maxwell® RSC 48.
Table 17. Clinical Comparison Results – Retrospective Study Results
Test Platform
Promega Maxwell® RSC 48 Positive %
Agreement (CI)1
Negative %
Agreement (CI)1
Result Positive Negative Inconclusive
QIAGEN EZ1®
Advanced XL
Positive 15 0 0
100.0
(79.6-100.0)
93.3
(70.2-98.9) Negative 0 14 0
Inconclusive 0 1 0
1 CI = 95% confidence interval
Disposal
Dispose of hazardous or biologically contaminated materials according to the practices of your
institution.
References
1. Ballew, H. C., et al. “Basic Laboratory Methods in Virology,” DHHS, Public Health Service 1975
(Revised 1981), Centers for Disease Control and Prevention, Atlanta, Georgia 30333.
2. Clinical Laboratory Standards Institute (CLSI), “Collection, Transport, Preparation and Storage of
Specimens for Molecular Methods: Proposed Guideline,” MM13-A
3. Lieber, M., et al. "A Continuous Tumor Cell Line from a Human Lung Carcinoma with Properties of
Type II Alveolar Epithelial Cells." International Journal of Cancer 1976, 17(1), 62-70.
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CDC-006-00019, Revision: 05 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 07/13/2020
Revision History
Revision # Effective Date Summary of Revisions
1 February 4, 2020 Original Instructions for Use
2 March 15, 2020 • Intended use update
• Removal of N3 primer and probe set from Diagnostic Panel
• Performance data update
• Addition of alternative nucleic acid extraction platforms
• Addition of acceptable alternatives to HSC and addition of
QIAGEN RUO extraction reagents
• Positive results no longer presumptive. No confirmation of
positive results required
3 March 30, 2020 • Addition of alternative enzyme master mix options
4 June 12, 2020 • Addition of MagNA Pure 24 extraction method
• Addition of performance data for the MagNA Pure 96
extraction method with SARS-CoV-2
• Addition of heat treatment alternative to specimen
extraction
• Addition of Roche and QIAGEN external lysis buffer
alternatives
• Acknowledgment of FDA policy permitting end users to
qualify alternative components without seeking an EUA or
EUA amendment
5 July 13, 2020 • Addition of Promega Maxwell® RSC 48 extraction method
• Update to in silico inclusivity analyses
Contact Information, Ordering, and Product Support
For technical and product support, contact the CDC Division of Viral Diseases directly.
Send email to: respvirus@cdc.gov
Note: If your laboratory is using reagents sourced from someone other than the CDC International
Reagent Resource, please refer to the manufacturer’s instructions provided with the commercial
materials.
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CDC-006-00019, Revision: 05 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 07/13/2020
Appendix A: Heat Treatment Alternative to Extraction
UltraPlex 1-Step ToughMix (4X)
This procedure is only for use by public health laboratories.
Purpose:
In response to a global shortage of nucleic acid extraction reagents causing significant delays in testing,
the CDC has investigated the use of a heat treatment method requiring minimal reagents as a specimen
processing alternative to nucleic acid extraction for use with the 2019-nCoV Real-Time RT-PCR Diagnostic
Panel.
Where possible, laboratories should use qualified RNA or total nucleic acid extraction methods for
processing of specimens for subsequent testing by the CDC 2019-nCoV Real-Time RT-PCR Diagnostic
Panel. Extraction removes inhibitory substances from specimens that could negatively impact PCR
performance.
This procedure for use of heat treatment for specimen processing is only recommended when a
shortage of qualified extraction reagents is a limiting factor in a laboratory’s ability to meet urgent
COVID-19 testing demand.
Precautions/Warnings/Limitations:
• CDC has evaluated this heat treatment process and has determined that this process is effective
for inactivation of SARS-CoV-2 in patient specimens.
• Performance was evaluated with only upper respiratory specimens. Heat treatment of lower
respiratory specimens for subsequent testing by the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel has not been evaluated.
• This procedure for heat treatment of specimens is only for use with the Quantabio UltraPlex 1-
Step ToughMix (4X).
• Heat treatment should only be conducted when a lab is ready to test the specimens by PCR.
Testing of heat-treated specimens must be conducted the same day.
Acceptable Specimens:
• Upper respiratory specimens
Note: Do not use heat treatment to process specimens that appear bloody or that contain
particulate matter. Such specimens should be extracted using a qualified RNA or TNA extraction
method prior to testing.
Materials Required (not provided):
• 70% ethanol
• 10% bleach, freshly prepared
• 96-well PCR reaction plates (Applied Biosystems catalog # 4346906, 4366932, 4346907, or
equivalent)
• Optical strip caps (Applied Biosystems 4323032, or equivalent)
• 1.5 mL Sarstedt tubes or equivalent
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CDC-006-00019, Revision: 05 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 07/13/2020
• Aerosol resistant micropipette tips
• Micropipettes
• 96-well cold block
• Cold blocks for 1.5 mL - 2.0 mL tubes
• Vortex mixer
• 96-well plate centrifuge or equivalent
• Thermal cycler or equivalent
• Class II Biological Safety Cabinet (BSC)
Procedure:
Sample Preparation
1) Decontaminate BSC with 10% bleach followed by 70% ethanol.
2) If samples are frozen, thaw on ice or at 4°C. Wipe the outside of the sample tube with 70%
ethanol. Place thawed sample on cold rack or ice in BSC.
3) Pulse vortex each sample and briefly spin down in a centrifuge to collect the liquid at the
bottom of the tube.
Heat Treatment
1) Place a thermal cycler in the BSC, turn on, and program for 95°C for 1 min followed by 4°C
hold.
2) Place a 96-well PCR plate onto a cold rack or ice in the BSC.
3) Transfer 100 µL of each sample to the 96-well PCR plate and securely cap each well using
optical strip caps.
NOTE: Ensure that an HSC extraction control is included in each batch run as required under
CLIA.
4) Place this 96-well PCR plate on the pre-heated thermal cycler and start run. Leave plate on
thermal cycler at 4°C, or place on ice or a cold block.
5) Remove plate and centrifuge for 1 minute at 500 x g to pellet cellular debris.
6) Place plate on a cold rack or ice and proceed to testing the supernatant by rRT-PCR.
7) Testing of heat-treated specimens must be conducted the same day heat treatment is
performed. For long term storage, keep the original specimen at ≤-70°C.
Special Testing Considerations for Heat Treated Specimens:
• Enzyme Master Mix
Testing of specimens that have been processed with heat treatment should be conducted
with the Quantabio UltraPlex 1-Step ToughMix (4X), which demonstrated the best
performance with heat treated specimens. PCR testing of heat-treated specimens should
follow the instructions in the main body of this Instructions for Use document.
• Resolution of Inconclusive and Invalid Results
Retesting of heat-treated specimens that generated an inconclusive or invalid result must
include extraction of the original specimen with a qualified RNA or total nucleic acid (TNA)
extraction method, if available. Do not re-test the heat-treated specimen material to resolve
inconclusive or invalid test results.
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CDC-006-00019, Revision: 05 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 07/13/2020
Verification:
CDC recommends performance of verification studies for the heat treatment method prior to diagnostic
use that includes side-by-side preparation of a panel of positive and negative clinical specimens using a
qualified extraction method and this heat treatment method with subsequent testing by the CDC 2019-
nCoV Real-Time RT-PCR Diagnostic Panel.
Performance Characteristics:
Quantabio UltraPlex 1-Step ToughMix (4X)
Limit of Detection Comparison
Serial dilutions of inactivated SARS-CoV-2 [SARS-CoV-2 USA-WA1/2020] were prepared in simulated
specimen material (human A549 cells suspended in viral transport medium). Each concentration was
prepared side-by-side five times by both EZ1 extraction and by heat treatment. Each extracted or heattreated sample was subsequently tested by the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel using
the Quantabio UltraPlex 1-Step ToughMix (4X) on the Applied Biosystems 7500 Fast Dx instrument.
Observed detection was similar between the two specimen preparation methods.
Table B1: UltraPlex Limit of Detection Comparison between QIAGEN EZ1 Advanced XL extraction and heat
treatment (95°C for 1 min) method – Summary Results
Enzyme Platform Parameter 2019-nCoV_N1 Assay 2019-nCoV_N2 Assay Observed
LoD1
Quantabio UltraPlex 1-
Step ToughMix (4X)
5 µL Template Addition
QIAGEN
EZ1
Advanced
XL
RNA copies/µL 101.0 100.5 100.0 10-0.5 10-1.0 101.0 100.5 100.0 10-0.5 10-1.0
100.5 # pos./total 5/5 5/5 4/5 4/5 3/5 5/5 5/5 5/5 2/5 2/5
Mean Ct2 34.11 34.59 NA NA NA 32.97 33.76 34.70 NA NA
Std. Deviation 0.75 0.99 NA NA NA 0.33 0.72 0.98 NA NA
Heat
Treatment
95°C
for 1 min
RNA copies/µL 101.0 100.5 100.0 10-0.5 10-1.0 101.0 100.5 100.0 10-0.5 10-1.0
100.5 # pos./total 5/5 5/5 4/5 5/5 1/5 5/5 5/5 4/5 2/5 1/5
Mean Ct2 33.41 34.32 NA 36.73 NA 33.45 35.25 NA NA NA
Std. Deviation 0.62 0.40 NA 0.82 NA 0.40 0.80 NA NA NA
1Concentration is presented in RNA copies/µL. The observed LoD is the lowest concentration where both assays showed 100%
positive detection.
2Mean Ct reported for dilutions that show 100% positivity. Calculations only include positive results.
NA = not applicable
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Clinical Comparison
A panel of 39 upper respiratory specimens were tested side-by-side using extraction with the Qiagen EZ1
extraction instrument and heat treatment. Extracted and heat-treated specimens were subsequently
tested with the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel using the Quantabio UltraPlex 1-Step
ToughMix (4X). Qualitative results were compared to demonstrate agreement.
Table B2: Clinical Comparison Results Summary – Heat Treatment versus QIAGEN EZ1 Advanced XL
Test Result Heat Treatment Total
Positive %
Agreement (CI)1
Negative %
Agreement (CI)1
Positive Inconclusive Negative
QIAGEN EZ1
Advanced XL
Positive 18 1 0 19
Inconclusive 0 0 0 0 94.7 (75.4-99.1) 100 (83.9-100)
Negative 0 0 20 20
Total 18 1 20 39
1 CI = 95% confidence interval
Questions and Comments:
If you have questions or comments about this procedure, please send by email to: respvirus@cdc.gov
Division of Viral Diseases / Respiratory Viruses Branch
CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel – Verification
Requirements
*** DO NOT DISCARD: Important product-specific information ***
Document #: CDC-006-00005 Revision #: 05 Effective Date: 07/13/2020 Page 1 of 4
CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel –
Verification Requirements
Please consult the following guidance from the Centers for Medicare & Medicaid Services
(CMS) regarding diagnostic tests under Emergency Use Authorization (EUA):
https://www.cms.gov/Medicare/Provider-Enrollment-andCertification/SurveyCertificationGenInfo/Policy-and-Memos-to-States-and-RegionsItems/QSO18-19-CLIA
INTENDED USE
The CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel is a realtime RT-PCR test intended for the qualitative detection of nucleic acid from the 2019-nCoV in
upper and lower respiratory specimens (such as nasopharyngeal or oropharyngeal swabs,
sputum, lower respiratory tract aspirates, bronchoalveolar lavage, and nasopharyngeal
wash/aspirate or nasal aspirate) collected from individuals who meet 2019-nCoV clinical
and/or epidemiological criteria (for example, clinical signs and symptoms associated with
2019-nCoV infection, contact with a probable or confirmed 2019-nCoV case, history of travel
to a geographic locations where 2019-nCoV cases were detected, or other epidemiologic links
for which 2019-nCoV testing may be indicated as part of a public health investigation). Testing
in the United States is limited to laboratories certified under the Clinical Laboratory
Improvement Amendments of 1988 (CLIA), 42 U.S.C. § 263a, to perform high complexity
tests.
Results are for the identification of 2019-nCoV RNA. The 2019-nCoV RNA is generally
detectable in upper and lower respiratory specimens during infection. Positive results are
indicative of active infection with 2019-nCoV but do not rule out bacterial infection or coinfection with other viruses. The agent detected may not be the definite cause of disease.
Laboratories within the United States and its territories are required to report all positive
results to the appropriate public health authorities.
Negative results do not preclude 2019-nCoV infection and should not be used as the sole basis
for treatment or other patient management decisions. Negative results must be combined with
clinical observations, patient history, and epidemiological information.
Testing with the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel is intended for use by
trained laboratory personnel who are proficient in performing real-time RT-PCR assays. The
CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel is only for use
under a Food and Drug Administration’s Emergency Use Authorization.
REQUIRED MATERIALS
The 2019 novel coronavirus positive control (nCoVPC) is provided with the CDC 2019-nCoV
Real-Time RT-PCR Diagnostic Panel and should be prepared according to the Instructions for
Use. The nCoVPC consists of an RNA transcript of the 2019-nCoV N gene as well as human
RNase P gene segment. nCoVPC will yield a positive result with the following primer and probe
sets: 2019-nCoV_N1, 2019-nCoV_N2, and RP.
Approximately 2 mL of an upper respiratory specimen (e.g. nasopharyngeal swab (NPS) in
transport media) are needed for testing. Specimens may be pooled if less than 2 mL of one
specimen is available.
Refer to CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel package insert (manufacturer
instructions) for additional reagents, materials, and instructions.
PRECAUTIONS
This reagent should be handled in an approved biosafety level 2 (BSL-2) handling area to
avoid contamination of laboratory equipment and reagents that could cause false positive
Division of Viral Diseases / Respiratory Viruses Branch
CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel – Verification
Requirements
*** DO NOT DISCARD: Important product-specific information ***
Document #: CDC-006-00005 Revision #: 05 Effective Date: Page 2 of 4
results. This product is an RNA transcript and is non-infectious. However, the nCoVPC should
be handled in accordance with Good Laboratory Practices.
Store reagent at appropriate temperatures (see Instructions for Use) and hold on ice when
thawed.
Please use standard precautions when handling respiratory specimens.
INSTRUCTIONS FOR PREPARING SAMPLES BEFORE EXTRACTION WITH THE
QIAamp® DSP VIRAL RNA MINI KIT OR THE QIAamp® VIRAL RNA MINI KIT
• Refer to the 2019-nCoV Real-Time RT-PCR Diagnostic Panel Instructions for Use for
reconstitution of the materials for use. RNA should be kept cold during preparation and use.
• Make a 1/10 dilution of nCoVPC by adding 5 µL of nCoVPC into 45 µL of nuclease-free water
or 10 mM Tris.
• Aliquot 560 µL of lysis buffer into each of nine tubes labeled 1-9.
• Add 140 µL of upper respiratory specimen (e.g. NPS in viral transport media) into each of
the nine labeled tubes with lysis buffer.
• To prepare samples at a moderate concentration, spike 14 µL of undiluted nCoVPC
(rehydrated as described in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Instructions for Use) into each tube labeled 1-3 containing lysis buffer and specimen.
• To prepare samples at a low concentration, spike 14 µL of 1/10 dilution of nCoVPC into
each tube labeled 4-6 containing lysis buffer and specimen.
• To prepare negative samples, spike 14 µL of nuclease-free water into each tube labeled 7-9
containing lysis buffer and specimen.
• Perform extractions of all nine samples according to the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel Instructions for Use.
INSTRUCTIONS FOR PREPARING SAMPLES BEFORE EXTRACTION WITH THE QIAGEN
EZ1® ADVANCED XL
• Refer to the 2019-nCoV Real-Time RT-PCR Diagnostic Panel Instructions for Use for
reconstitution of the materials for use. RNA should be kept cold during preparation and use.
• Make a 1/10 dilution of nCoVPC by adding 5 µL of nCoVPC into 45 µL of nuclease-free water
or 10 mM Tris.
• Aliquot 280 µL of lysis buffer into each of nine tubes labeled 1-9.
• Add 120 µL of upper respiratory specimen (e.g. NPS in viral transport media) into each of
the nine labeled tubes with lysis buffer.
• To prepare samples at a moderate concentration, spike 12 µL of undiluted nCoVPC
(rehydrated as described in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Instructions for Use) into each tube labeled 1-3 containing lysis buffer and specimen.
• To prepare samples at a low concentration, spike 12 µL of 1/10 dilution of nCoVPC into
each tube labeled 4-6 containing lysis buffer and specimen.
• To prepare negative samples, spike 12 µL of nuclease-free water into each tube labeled 7-9
containing lysis buffer and specimen.
• Perform extractions of all nine samples according to the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel Instructions for Use.
INSTRUCTIONS FOR PREPARING SAMPLES BEFORE EXTRACTION WITH THE ROCHE
MagNA PURE TOTAL NUCLEIC ACID KIT OR THE ROCHE MagNA PURE NUCLEIC ACID
ISOLATION KIT I
• Refer to the 2019-nCoV Real-Time RT-PCR Diagnostic Panel Instructions for Use for
reconstitution of the materials for use. RNA should be kept cold during preparation and use.
• Make a 1/10 dilution of nCoVPC by adding 5 µL of nCoVPC into 45 µL of nuclease-free water
or 10 mM Tris.
• Aliquot 300 µL of lysis buffer into each of nine tubes labeled 1-9.
• Add 100 µL of upper respiratory specimen (e.g. NPS in viral transport media) into each of
the nine labeled tubes with lysis buffer.
Division of Viral Diseases / Respiratory Viruses Branch
CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel – Verification
Requirements
*** DO NOT DISCARD: Important product-specific information ***
Document #: CDC-006-00005 Revision #: 05 Effective Date: Page 3 of 4
• To prepare samples at a moderate concentration, spike 12 µL of undiluted nCoVPC
(rehydrated as described in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Instructions for Use) into each tube labeled 1-3 containing lysis buffer and specimen.
• To prepare samples at a low concentration, spike 12 µL of 1/10 dilution of nCoVPC into
each tube labeled 4-6 containing lysis buffer and specimen.
• To prepare negative samples, spike 12 µL of nuclease-free water into each tube labeled 7-9
containing lysis buffer and specimen.
• Perform extractions of all nine samples according to the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel Instructions for Use.
INSTRUCTIONS FOR PREPARING SAMPLES BEFORE EXTRACTION WITH THE ROCHE
MagNA PURE 24 AND TOTAL NUCLEIC ACID ISOLATION KIT
• Refer to the 2019-nCoV Real-Time RT-PCR Diagnostic Panel Instructions for Use for
reconstitution of the materials for use. RNA should be kept cold during preparation and use.
• Make a 1/10 dilution of nCoVPC by adding 5 µL of nCoVPC into 45 µL of nuclease-free water
or 10 mM Tris.
• Aliquot 400 µL of lysis buffer into each of nine tubes labeled 1-9.
• Add 100 µL of upper respiratory specimen (e.g. NPS in viral transport media) into each of
the nine labeled tubes with lysis buffer.
• To prepare samples at a moderate concentration, spike 12 µL of undiluted nCoVPC
(rehydrated as described in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Instructions for Use) into each tube labeled 1-3 containing lysis buffer and specimen.
• To prepare samples at a low concentration, spike 12 µL of 1/10 dilution of nCoVPC into
each tube labeled 4-6 containing lysis buffer and specimen.
• To prepare negative samples, spike 12 µL of nuclease-free water into each tube labeled 7-9
containing lysis buffer and specimen.
• Perform extractions of all nine samples according to the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel Instructions for Use.
INSTRUCTIONS FOR PREPARING SAMPLES BEFORE EXTRACTION WITH THE ROCHE
MagNA PURE 96 DNA AND VIRAL NA SMALL VOLUME KIT
• Refer to the 2019-nCoV Real-Time RT-PCR Diagnostic Panel Instructions for Use for
reconstitution of the materials for use. RNA should be kept cold during preparation and use.
• Make a 1/10 dilution of nCoVPC by adding 5 µL of nCoVPC into 45 µL of nuclease-free water
or 10 mM Tris.
• Aliquot 350 µL of lysis buffer into each of nine tubes labeled 1-9.
• Add 100 µL of upper respiratory specimen (e.g. NPS in viral transport media) into each of
the nine labeled tubes with lysis buffer.
• To prepare samples at a moderate concentration, spike 12 µL of undiluted nCoVPC
(rehydrated as described in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Instructions for Use) into each tube labeled 1-3 containing lysis buffer and specimen.
• To prepare samples at a low concentration, spike 12 µL of 1/10 dilution of nCoVPC into
each tube labeled 4-6 containing lysis buffer and specimen.
• To prepare negative samples, spike 12 µL of nuclease-free water into each tube labeled 7-9
containing lysis buffer and specimen.
• Perform extractions of all nine samples according to the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel Instructions for Use.
INSTRUCTIONS FOR PREPARING SAMPLES BEFORE EXTRACTION WITH THE
PROMEGA MAXWELL® RSC 48
• Refer to the 2019-nCoV Real-Time RT-PCR Diagnostic Panel Instructions for Use for
reconstitution of the materials for use. RNA should be kept cold during preparation and use.
• Make a 1/10 dilution of nCoVPC by adding 5 µL of nCoVPC into 45 µL of nuclease-free water
or 10 mM Tris.
• Aliquot 330 µL of lysis buffer (300 µL of lysis buffer + 30 µL Proteinase K, included in the
kit) into each of nine tubes labeled 1-9.
Division of Viral Diseases / Respiratory Viruses Branch
CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel – Verification
Requirements
*** DO NOT DISCARD: Important product-specific information ***
Document #: CDC-006-00005 Revision #: 05 Effective Date: Page 4 of 4
• Add 120 µL of upper respiratory specimen (e.g. NPS in viral transport media) into each of
the nine labeled tubes with lysis buffer.
• To prepare samples at a moderate concentration, spike 12 µL of undiluted nCoVPC
(rehydrated as described in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Instructions for Use) into each tube labeled 1-3 containing lysis buffer and specimen.
• To prepare samples at a low concentration, spike 12 µL of 1/10 dilution of nCoVPC into
each tube labeled 4-6 containing lysis buffer and specimen.
• To prepare negative samples, spike 12 µL of nuclease-free water into each tube labeled 7-9
containing lysis buffer and specimen.
• Perform extractions of all nine samples according to the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel Instructions for Use.
INSTRUCTIONS FOR PREPARING SAMPLES BEFORE EXTRACTION WITH THE
BIOMÉRIEUX NucliSENS easyMAG OR THE BIOMÉRIEUX EMAG
• Refer to the 2019-nCoV Real-Time RT-PCR Diagnostic Panel Instructions for Use for
reconstitution of the materials for use. RNA should be kept cold during preparation and use.
• Make a 1/10 dilution of nCoVPC by adding 5 µL of nCoVPC into 45 µL of nuclease-free water
or 10 mM Tris.
• Aliquot 1000 μL or 2000 µL of pre-aliquoted easyMAG lysis buffer into each of nine tubes
labeled 1-9 for the easyMAG or eMAG, respectively.
• Add 100 µL of upper respiratory specimen (e.g. NPS in viral transport media) into each of
the nine labeled tubes with lysis buffer.
• To prepare samples at a moderate concentration, spike 12 µL of undiluted nCoVPC
(rehydrated as described in the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel
Instructions for Use) into each tube labeled 1-3 containing lysis buffer and specimen.
• To prepare samples at a low concentration, spike 12 µL of 1/10 dilution of nCoVPC into
each tube labeled 4-6 containing lysis buffer and specimen.
• To prepare negative samples, spike 12 µL of nuclease-free water into each tube labeled 7-9
containing lysis buffer and specimen.
• Perform extractions of all nine samples according to the CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel Instructions for Use.
PROCEDURE
Follow the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel Instructions for Use for testing
the nine extracted samples at least once.
EXPECTED RESULTS
Moderate nCoVPC samples should be positive for 2019-nCoV.
Low nCoVPC samples should be positive for 2019-nCoV.
Negative upper respiratory samples should be negative for 2019-nCoV.
≥90% of test results should be in agreement with the expected results. If test results are
<90% in agreement with expected results, contact CDC at respvirus@cdc.gov.
QUESTIONS
Please send questions or comments by email to respvirus@cdc.gov.
DISTRIBUTION
Distributed to qualified laboratories by Centers for Disease Control and Prevention, 1600
Clifton Road, Atlanta, GA, 30329 USA

page list

23 Oct 2020

CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel