CDC 2019-Novel Coronavirus (2019-nCoV) 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

Centers for Disease Control and Prevention Division of Viral Diseases
1600 Clifton Rd NE
Atlanta GA 30329

CDC-006-00019, Revision: 05 CDC/DDID/NCIRD/ Division of Viral Diseases Effective: 07/13/2020

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

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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:

Positive Control (either of the following products are acceptable) Catalog #2019-nCoVEUA-01 Diagnostic Panel Box #2:

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Catalog #VTC-04 CDC 2019-nCoV Positive Control (nCoVPC)

Materials Required (But Not Provided)

Human Specimen Control (HSC)

Acceptable alternatives to HSC:

• Negative human specimen material: Laboratories may prepare a volume of human specimenmaterial (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

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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.

1Equivalence 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 SARS- CoV-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 MagNAPure 24, Roche MagNA Pure 96, Promega Maxwell® RSC 48, Roche MagNA Pure LC, RocheMagNA 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 AWAYTM (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, without9
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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 underan EUA for use by laboratories certified under CLIA, 42 U.S.C. § 263a, to perform highcomplexity tests.
  • This test has been authorized only for the detection of nucleic acid from SARSCoV-2, notfor any other viruses or pathogens.
  • This test is only authorized for the duration of the declaration that circumstances existjustifying 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. § 360bbb- 3(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 notsubstitute 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 essentialto minimize the risk of cross-contamination between samples and the inadvertent10
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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 extractednucleic acids.
•   Wear a clean lab coat and powder-free disposable gloves (not previously worn) whensetting 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 andmaintained 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, DNAZapTM, or RNase AWAYTM to minimize risk ofnucleic 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 ensurestability.
• Dispose of unused kit reagents and human specimens according to local, state, and federalregulations.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 coldblock 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 preparationand 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 orDacron®, 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 requirementsfor 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) 639-3931The 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. 1)  Upon receipt, store dried primers and probes at 2-8°C.
2. 2)  Precautions: These reagents should only be handled in a clean area and stored at appropriatetemperatures (see below) in the dark. Freeze-thaw cycles should be avoided. Maintain coldwhen thawed.
3. 3)  Using aseptic technique, suspend dried reagents in 1.5 mL of nuclease-free water and allowto rehydrate for 15 min at room temperature in the dark.
4. 4)  Mix gently and aliquot primers/probe in 300 μL volumes into 5 pre-labeled tubes. Store asingle, 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).13

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2019-nCoV Positive Control (nCoVPC) Preparation:

1. 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. 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. 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. 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. 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 DNAzapTM, or RNase AWAYTM 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

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. 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. 2)  Mix buffer, enzyme, and primer/probes by inversion 5 times.
3. 3)  Centrifuge reagents and primers/probes for 5 seconds to collect contents at the bottom ofthe tube, and then place the tube in a cold rack.
4. 4)  Label one 1.5 mL microcentrifuge tube for each primer/probe set.
5. 5)  Determine the number of reactions (N) to set up per assay. It is necessary to make excessreaction 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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Reaction Master Mix and Plate Set Up

Thermofisher TaqPathTM 1-Step RT-qPCR Master Mix

Promega GoTaq® Probe 1- Step RT-qPCR System

Quantabio qScript XLT One-Step RT-qPCR ToughMix

Quantabio UltraPlex 1-Step ToughMix (4X)

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8. 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. 9)  Centrifuge for 5 seconds to collect contents at the bottom of the tube, and then place the tube in a cold rack.
10. 10)  Set up reaction strip tubes or plates in a 96-well cooler rack.
11. 11)  Dispense 15 μL of each master mix into the appropriate wells going across the row as shownbelow (Figure 1):

Figure 1: Example of Reaction Master Mix Plate Set-Up

12. 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. 13)  Pipette 5 μL of nuclease-free water into the NTC sample wells (Figure 2, column 1). Securely cap NTC wells before proceeding.
14. 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. 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. 9)  Cover the entire reaction plate and move the reaction plate to the positive template control handling area.

Assay Control Addition

1. 1)  Pipette 5 μL of nCoVPC RNA to the sample wells of column 12 (Figure 2). Securely cap wellsafter addition of the control RNA.NOTE: If using 8-tube strips, label the TAB of each strip to indicate sample position. DO NOTLABEL THE TOPS OF THE REACTION TUBES!
2. 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

aReplace 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. 1)  Launch the Applied Biosystems 7500 Fast Dx Real-time PCR Instrument by double clicking on theApplied Biosystems 7500 Fast Dx System icon on the desktop.
2. 2)  A new window should appear, select Create New Document from the menu.

Figure 3. New Document Wizard Window

3. 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 Documentd. Run Mode: Standard 7500 e. Operator: Your Name
   f. Comments: SDS v1.4
   g. Plate Name: Your Choice
4. 4)  After making selections click Next at the bottom of the window.20

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Make sure to change Run Mode to STANDARD 7500

Figure 4. Creating New Detectors

NOTE: ROX is the default passive reference. This will be changed to “none” in step 12.

5. 5)  After selecting next, the Select Detectors screen (Figure 4) will appear.
6. 6)  Click the New Detector button (see Figure 4).
7. 7)  The New Detector window will appear (Figure 5). A new detector will need to be defined foreach 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 a. b. c. d. e.

by creating the N1 Detector. Include the following: Name: N1
Description: leave blank
Reporter Dye: FAM

Quencher Dye: (none)
Color: to change the color of the detector indicator do the following:

⇒ Clickonthecolorsquaretorevealthecolorchart
⇒ Select a color by clicking on one of the squares
⇒ AfterselectingacolorclickOKtoreturntotheNewDetectorscreen

Click the OK button of the New Detector screen to return to the screen shown in Figure 4.

f.
9) Repeat step 6-8 for each target in the panel.

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10. 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. 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. 1)  After detectors have been created and assigned, proceed to instrument set up.
2. 2)  Select the Instrument tab to define thermal cycling conditions.
3. 3)  Modify the thermal cycling conditions as follows (Figure 12):Thermofisher TaqPathTM 1-Step RT-qPCR Master Mix, CG
   1. InStage1,Setto2minat25°C;1Rep.
   2. In Stage 2, Set to 15 min at 50°C; 1 Rep.
   3. InStage3,Setto2minat95°C,1Rep.
   4. InStage4,Step1setto3secat95°C.
   5. In Stage 4, Step 2 set to 30 sec at 55.0°C.
   6. In Stage 4, Reps should be set to 45.
   7. Under Settings (Figure 12), bottom left-hand box, change volume to 20 μL.
   8. Under Settings, Run Mode selection should be Standard 7500.
   9. Step 2 of Stage 4 should be highlighted in yellow to indicate data collection (see Figure12).

   OR

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Quantabio qScriptTM XLT One-Step RT-qPCR ToughMix or UltraPlex 1-Step ToughMix (4X)

1. In Stage 1, Set to 10 min at 50°C; 1 Rep.
2. InStage2,Setto3minat95°C,1Rep.
3. InStage3,Step1setto3secat95°C.
4. In Stage 3, Step 2 set to 30 sec at 55.0°C.
5. In Stage 3, Reps should be set to 45.
6. Under Settings (Figure 12), bottom left-hand box, change volume to 20 μL.
7. Under Settings, Run Mode selection should be Standard 7500.
8. Step 2 of Stage 3 should be highlighted in yellow to indicate data collection (see Figure12).

OR
Promega GoTaq® Probe 1-Step RT-qPCR System

1. In Stage 1, Set to 15 min at 45°C; 1 Rep.
2. InStage2,Setto2minat95°C,1Rep.
3. InStage3,Step1setto3secat95°C.
4. In Stage 3, Step 2 set to 30 sec at 55.0°C.
5. In Stage 3, Reps should be set to 45.
6. Under Settings (Figure 12), bottom left-hand box, change volume to 20 μL.
7. Under Settings, Run Mode selection should be Standard 7500.
8. Step 2 of Stage 3 should be highlighted in yellow to indicate data collection (see Figure12).

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Figure 12. Instrument Window