WHO Emergency Use Assessment Coronavirus disease …...The Multiple Real-Time PCR Kit for Detection...

EUL-0491-187-00 WHO EUL Public Report July 2020, version 1.0

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WHO Emergency Use Assessment Coronavirus disease (COVID-19) IVDs PUBLIC REPORT

Product: Multiple Real-Time PCR Kit for Detection of

2019-CoV EUL Number: EUL-0491-187-00

Outcome: Accepted

The EUL process is intended to expedite the availability of in vitro diagnostics needed in public health emergency situations and to assist interested UN procurement agencies and Member States in determining the acceptability of using specific products in the context of a Public Health Emergency of International Concern (PHEIC), based on an essential set of available quality, safety and performance data. The EUL procedure includes the following:

• Quality Management Systems Review and Plan for Post-Market Surveillance: desk-top review of the manufacturer’s Quality Management System documentation and specific manufacturing documents;

• Product Dossier Review: assessment of the documentary evidence of safety and performance.

Multiple Real-Time PCR Kit for Detection of 2019-CoV with product code CT8233-48T, RoW regulatory version manufactured by Beijing Applied Biological Technologies Co. Ltd., (XABT), Room A204, B304, D203, D203-1, No.29 Shengmingyuan Road, Science Park, Changping District, Beijing, 102206, China, was listed as eligible for WHO procurement on 19 May 2020. Intended use: According to the claim of intended use from Beijing Applied Biological Technologies Co. Ltd., (XABT), “the Multiple Real-Time PCR Kit for Detection of 2019-nCoV is used for qualitative in vitro test of SARS-CoV-2 ORF1ab gene, N gene and E gene in oropharyngeal swab, nasopharyngeal swab and sputum in suspected cases and suspected cluster cases of infection with SARS-CoV-2 in aiding the diagnosis of SARS-CoV-2 infection. The Multiple Real-Time PCR Kit for Detection of 2019-nCoV is an in vitro diagnostic medical device, based on real time RT-PCR technology utilizing reverse-transcriptase (RT) reaction to convert RNA into complementary DNA (cDNA). Reactive results should be confirmed by details of confirmatory step which have been introduced in the Interpretation of Results. The assay is used to detect, define or differentiate a condition or risk factor of interest. The assay is manually operated and to be used with the RNA extraction kits and PCR instruments. The assay is for use by a laboratory professionally trained to use real time PCR in a laboratory setting.”


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Specimen type(s) that were validated: Oropharyngeal and nasopharyngeal swabs and sputum specimens. Test kit contents:

Component 48 tests (product code CT8233-48T)

Nuclease-free water 1 vial x 1 000 μL Nucleic acid amplification reaction solution 1 vial x 960μL 20×Reverse transcriptase 1 vial x 96μL 10×O/N reaction solution(A) 1 vial x 96μL 10×E reaction solution(B) 1 vial x 96μL Internal control 1 vial x 480μL Positive control（ O/N/E（ 1 vial x 480μL Negative control 1 vial x 480μL User manual -

Items required but not provided: Extraction/Purification: Extraction reagents:

• Nucleic acid extraction Kit (XABT, Cat. # CN8033); • QIAamp Viral RNA Mini Kit (Qiagen, Cat. # 52904 or 52906); • PURELINK VIRAL RNA/DNA KIT (Invitrogen, Cat. #12280050); • High Pure Viral RNA Kit (Roche, Cat. # 11858882001).

General laboratory equipment and consumables:

• Freezer (-20oC±5oC, -80oC) and refrigerator (2oC ~ 8oC) • Vortex • Microcentrifuge (with rotor for 1.5mL and 2mL tubes) • RNase-free microtubes • Heating block or water bath for lysis of specimens at 56oC • Tube racks • Class II biosafety cabinet (or glove box) • Personal protective equipment: Powder-free gloves, lab coat, eye protection, etc. • Pipets and pipette tips (To prevent cross-contamination, we strongly recommend the

use of pipette • tips with aerosol barriers) • Ethanol (96 ~ 100%)


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Amplification and detection instruments: • Nucleic acid extraction Kit (XABT, Cat. # CN8033) • QIAamp Viral RNA Mini Kit (Qiagen, Cat. # 52904 or 52906) • PURELINK VIRAL RNA/DNA KIT (Invitrogen, Cat. #12280050) • High Pure Viral RNA Kit (Roche, Cat. # 11858882001).

Storage: Store all reagents below -18°C in dark. Shelf-life upon manufacture: 6 months, real-time stability study is ongoing. Warnings/limitations: Refer to the instructions for use (IFU) Product dossier assessment

Beijing Applied Biological Technologies Co. Ltd., (XABT) submitted a product dossier for the Multiple Real-Time PCR Kit for Detection of 2019-nCoV for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as per the “Instructions for Submission Requirements: In vitro diagnostics (IVDs) Detecting SARS-CoV-2 Nucleic Acid (PQDx_0347 version 4)”. The information (data and documentation) submitted in the product dossier was reviewed by WHO staff and external technical experts (assessors) appointed by WHO. Post listing Commitments for EUL:

1. As a commitment to listing, the manufacturer is required to participate in the WHO collaborative study for the assessment of the suitability of an interim standard for SARS-CoV-2 virus nucleic acid amplification tests.

2. As a commitment to listing, the manufacturer is required to provide the operating temperature study results when complete.

Risk benefit assessment conclusion: acceptable.


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Quality Management Systems Review To establish the eligibility for WHO procurement, Beijing Applied Biological Technologies Co. Ltd., (XABT) was asked to provide up-to-date information about the status of their quality management system. Based on the review of the submitted quality management system documentation by WHO staff, it was established that sufficient information was provided by Beijing Applied Biological Technologies Co. Ltd., (XABT) to fulfil the requirements described in the “Instructions for Submission Requirements: In vitro diagnostics (IVDs) Detecting SARS-CoV-2 Nucleic Acid (PQDx_ 347 version 4)”. Quality management documentation assessment conclusion: acceptable. Plan for Post-Market Surveillance Post-market surveillance, including monitoring all customer feedback, detecting and acting on adverse events, product problems, non-conforming goods and processes is a critical component of minimizing potential harm of an IVD listed for emergency use. The following post-EUL activities are required to maintain the EUL listing status: 1. Notification to WHO of any planned changes to a EUL product, in accordance with “WHO procedure for changes to a WHO prequalified in vitro diagnostic” (document number PQDx_121); and 2. Post-market surveillance activities, in accordance with “WHO guidance on post-market surveillance of in vitro diagnostics” (ISBN 978 92 4 150921 3). Beijing Applied Biological Technologies Co. Ltd., (XABT) is also required to submit an annual report that details sales data and all categories of complaints in a summarized form. There are certain categories of complaints and changes to the product that must be notified immediately to WHO, as per the above-mentioned documents. The manufacturer has committed to ensure that post-emergency use listing safety, quality and performance monitoring activities are in place which are in accordance with WHO guidance “WHO guidance on post-market surveillance of in vitro diagnostics”.1 Scope and duration of procurement eligibility The Multiple Real-Time PCR Kit for Detection of 2019-nCoV for detecting 2019-nCoV with product code MFG030011 manufactured by Beijing Applied Biological Technologies Co. Ltd., (XABT) is considered to be eligible for WHO procurement for 12 months from the day of listing.

1 Available on the web page https://www.who.int/diagnostics_laboratory/postmarket/en/

https://www.who.int/diagnostics_laboratory/postmarket/en/

https://www.who.int/diagnostics_laboratory/postmarket/en/


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The assay may be used for the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This listing does not infer that the product meets WHO prequalification requirements and does not mean that the product is listed as WHO prequalified.

As part of the on-going requirements for listing as eligible for WHO procurement, Beijing Applied Biological Technologies Co. Ltd., (XABT) must engage in post-market surveillance activities to ensure that the product continues to meet safety, quality and performance requirements. Beijing Applied Biological Technologies Co. Ltd., (XABT) is required to notify WHO of any complaints, including adverse events related to the use of the product within 7 days.

WHO reserves the right to rescind eligibility for WHO procurement, if additional information on the safety, quality, performance during post-market surveillance activities, and if new data becomes available to WHO that changes the risk benefit balance.


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Labelling

1.0 Labels

2.0 Instructions for Use (IFU)


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1.0 Labels

Multiple Real-Time PCR Kit for Detection of 2019-nCoV ZCHS-C-YF-JC19-11 V0.1

Page 1 of4

Packaging Sample Design of Multiple Real-Time PCR Kit for Detection of

2019-nCoV

Objective: To standardize the design of English packaging labels for Multiple

Real-Time PCR Kit for Detection of 2019-nCoV.

Scope: Designed for English packaging labels of our company's Multiple Real-Time

PCR Kit for Detection of 2019-nCoV.

Content：

1. Outer box and lining：

Packing specification: 60×45×65mm.

Lining specification: 60×45mm; The diameter error of lining is ±1mm.

2. Sample box label：


Page 2 of4

3. Sample reagent bottle label：


Page 3 of4


Page 4 of4


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2.0 Instructions for use2

2 English version of the IFU was the one that was assessed by WHO. It is the responsibility of the manufacturer to ensure correct translation into other languages.

Beijing Applied Biological Technologies Co., Ltd.

Multiple Real-Time PCR Kit for Detection of 2019-nCoVRevision No.: ABT/WHO-JC19-01 V1.0, Issue Date: March 19, 2020

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Revision No.: ABT/WHO-JC19-01 V2.0

Issue Date: June 18, 2020

XABT SARS-CoV-2Multiple Real-Time PCR Kit for Detection of

2019-nCoV

Instruction for Use

For In Vitro Diagnostic Use

CT8233-48T

For use with ABI 7500; Bio-Rad CFX96; Roche LightCycler 480 II Instruments


Registered Address: Room A204, 29 Science ParkRoad, Zhongguancun Life Science Park, ChangpingDistrict, Beijing 102206, P.R.China.

Manufacture Address: Room A204, B304, D203,D203-1, 29 Science Park Road, Zhongguancun LifeScience Park, Changping District, Beijing 102206,P.R.China.

Tel:+86-10-80727800

Email：[email protected]

Website：http://www.x-abt.com/en/

Tel:+86-10-80727800



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CONTENTS

Instruction for Use.........................................................................................................................1General Information............................................................................................................................ 3

1. Intended Use............................................................................................................................. 32. Principle of Real Time RT-PCR...................................................................................................33. Product Description...................................................................................................................44. Warnings and Precautions........................................................................................................ 45. Kit Contents...............................................................................................................................66. Storage...................................................................................................................................... 67. Specimen Requirements........................................................................................................... 67.1 Specimen Types.......................................................................................................................77.2 Specimen Storage....................................................................................................................77.3 Shipment................................................................................................................................. 88. RNA Extraction Procedure for Specimens and Controls........................................................... 88.1 Test Preparation......................................................................................................................88.2 RNA Isolation...........................................................................................................................98.3 Test Method..........................................................................................................................18Instrument Specific Instructions................................................................................................. 209. Set Up...................................................................................................................................... 20

Interpretation of Results................................................................................................................... 3210. Interpretation of Controls and Clinical Specimens............................................................... 3210.1 Controls............................................................................................................................... 3210.2 Specimen.............................................................................................................................3211. Limitations.............................................................................................................................3412. Analytical Performance Characteristics................................................................................ 3512.1 Limit of Detection................................................................................................................3512.2 Analytical Specificity............................................................................................................3512.3 Precision..............................................................................................................................3713. Clinical Evaluation................................................................................................................. 38

References..........................................................................................................................................40



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General Information

1. Intended Use

The Multiple Real-Time PCR Kit for Detection of 2019-nCoV is used for qualitative in vitro test of SARS-CoV-2 ORF1ab gene, N gene and E gene in oropharyngeal swab, nasopharyngeal swab and sputum in suspected

cases and suspected cluster cases of infection with SARS-CoV-2 in aiding the diagnosis of SARS-CoV-2infection.

The Multiple Real-Time PCR Kit for Detection of 2019-nCoV is an in vitro diagnostic medical device, basedon real time RT-PCR technology utilizing reverse-transcriptase (RT) reaction to convert RNA into

complementary DNA (cDNA). Reactive results should be confirmed by details of confirmatory step whichhave been introduced in the Interpretation of Results. The assay is used to detect, define or differentiate acondition or risk factor of interest.

The assay is manually operated and to be used with the RNA extraction kits and PCR instruments. The assay

is for use by a laboratory professionally trained to use real time PCR in a laboratory setting.

The use of RT-PCR method shortens the testing window, which is conducive to earlier identification andtreatment of infection.

2. Principle of Real Time RT-PCR

Real time reverse transcription polymerase chain reaction (Real Time RT-PCR) is used when the starting

material is RNA. In this method, RNA is first transcribed into complementary DNA (cDNA) by reversetranscriptase from total RNA. The cDNA is then used as a template for the real time PCR.

PCR is a process for amplifying target DNA sequence with thermophilic DNA polymerase. It involves threesteps: melting (denaturing of the DNA duplex at a high temperature to yield single stranded DNA),

annealing (primers anneal to the single stranded target sequence) and elongation (DNA polymeraseextends the primers by adding dNTPs to the phosphate backbone). These steps complete one PCR cycle,

and the cycle repeats until a sufficient DNA concentration is reached.

Real time PCR is a PCR with the advantage of detecting the amount of DNA formed after each cycle with a

fluorescently-tagged oligonucleotide probe. The probe is complementary to the target sequence beingamplified. A fluorophore attached to the 5’ end of the probe and a quencher dye attached to the 3’ end.The fluorescence emitted from fluorophore is quenched by the quencher dye and so no fluorescence can

be detected in the absence of target sequence. During each real time PCR cycle the probe hybridizes to its



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target sequence, downstream from a PCR primer. As DNA polymerase extends the primer, it encountersand hydrolyzes the probe from the 5’ end, releasing the fluorophore from the probe. The fluorophore isexcited and its emission is no longer quenched and there is an increase in fluorescence which can be

detected by real time PCR instrument. Testing data is generated, collected and analyzed by specializedsoftware.

3. Product Description

Coronaviruses belong to Nidovirales and Coronaviridae. According to serotypes and genomic characteristics,

coronaviruses are divided into four genera: α, β, γ and δ. Among them, there are 7 coronavirus infectinghuman beings. 229E and NL63 belong to α genus; OC43 and HKU1, middle east respiratory syndrome

related coronavirus (MERSr-CoV), severe acute respiratory syndrome associated coronavirus (SARSr-CoV),and the novel coronaviruses belong to the β genus. COVID-19 is an acute respiratory infectious disease.

People are generally susceptible. Currently, the patients infected by the novel coronavirus are the mainsource of infection; asymptomatic infected people can also be an infectious source. Based on the current

epidemiological investigation, the incubation period is 1 to 14 days, mostly 3 to 7 days. The mainmanifestations include fever, fatigue and dry cough. Nasal congestion, runny nose, sore throat, myalgia anddiarrhea are found in a few cases. It may rapidly develop into severe pneumonia, respiratory failure, acute

respiratory distress syndrome, septic shock, multiple organ failure, severe acid-base disturbance, and even

endanger life. The 2019-nCoV is mainly transmitted through direct contact with secretions or aerosol anddroplets. Laboratory detection methods for 2019-nCoV mainly include virus isolation, nucleic acid detectionand so on (Technical Guidelines for Covid-19 Laboratory Testing (Fifth Edition)).

Multiple Real-Time PCR Kit for Detection of 2019-nCoV contains a specialized ready-to-use one step realtime RT-PCR system for the detection of ORF1ab, N and E genes specifically of SARS-CoV-2, which includes

reverse transcription (RT) for the transcription of SARS-CoV-2 RNA into cDNA and real time PCR for theamplification and detection of cDNA from SARS-CoV-2 RNA.

A positive control, a negative control and an internal control (IC) are included in this kit to identify falsenegative results, false positive results, low extraction efficiency and possible PCR inhibitors.

Experimental operators should get professional trainings in gene amplification or molecular biology testingfield, have relevant qualifications, and the laboratory should have reasonable biological safety precautions

and protective procedures.

4. Warnings and Precautions Read this user manual carefully before using the kit.

Before first use please check the product and its components for integrity according to “5. Kit



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Contents” below, completeness, correct labeling and that the kit components are frozen uponarrival.

Use separated and segregated working areas for (i) specimen preparation, (ii) reaction setup and (iii)

amplification/detection activities. The workflow in the laboratory should proceed in unidirectionalmanner. Always wear disposable gloves in each area and change them before entering a different

area.

Dedicate supplies and equipment to the separate working areas and do not move them from one

area to another.

Specimens should always be treated as infectious and/or biohazardous in accordance with safe

laboratory procedures.

Avoid microbial and nuclease (DNase/RNase) contamination of the specimens and the components

of the kit.

Store positive and/or potentially positive material separated from all other components of the kit.

Use of this product is limited to personnel specially instructed and trained in the techniques of real

time PCR and in vitro diagnostic procedures.

Wear protective disposable powder-free gloves, a laboratory coat and eye protection when

handling specimens.

Always use DNase/RNase-free disposable pipette tips with aerosol barriers.

Always wear protective disposable powder-free gloves when handling kit components.

Additional controls may be tested according to guidelines or requirements of local, state and/or

federal regulations or accrediting organizations.

Do not open the reaction tubes/plates post amplification, to avoid contamination with amplicons.

Do not autoclave reaction tubes after the PCR, since this will not degrade the amplified nucleic acidand will bear the risk to contaminate the laboratory area.

Discard specimen and assay waste according to your local safety regulations.

Do not use components of the kit that have passed the expiration date.

Frozen reagents should be thawed completely at room temperature before commencing testing.



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5. Kit Contents

Reagents Quantity Sufficient for 48 Tests Color Coding

Nuclease-free water 1 vial, 1000μL whiteNucleic acid amplification reaction solution 1 vial, 960μL green

20×Reverse transcriptase 1 vial, 96μL transparent10×O/N reaction solution(A) 1 vial, 96μL orange10×E reaction solution(B) 1 vial, 96μL blue

Internal control 1 vial, 480μL purple

Positive control（O/N/E） 1 vial, 480μL redNegative control 1 vial, 480μL yellowUser Manual 1 -

Control materials Negative Control is physiological saline that will serve as an external negative specimen during RNA

extraction procedure. It should be used whenever RNA extraction procedure takes place.

Positive Control (O/N/E) is a pseudovirus containing ORF1ab, N and E gene fragments of SARS-CoV-2 that will serve as an external positive control during RNA extraction procedure. The SARS-CoV-2

RNA fragment in pseudovirus is designed to cover the target sequence to react with the real time

RT-PCR reagents in this kit to indicate whether the real time RT-PCR worked. It should be usedwhenever RNA extraction procedure takes place.

Internal Control is a pseudovirus containing non-target RNA fragment that will be added into the

specimen before RNA extraction procedure as internal control to evaluate RNA extraction efficiencyand identify possible PCR inhibitors. The RNA fragment in pseudovirus will be amplified by the

primers used to amplify target sequence, but it will be detected by another probe. Internal Controlshould be used whenever RNA extraction procedure takes place.

6. Storage All reagents should be stored at -20oC±5oC.

All reagents should be used before expiration date indicated on kit.

Repeated thaw-freeze for more than 7 times should be avoided as this may reduce the sensitivity

of the assay.

7. Specimen Requirements



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7.1 Specimen Types

Human oropharyngeal swab, nasopharyngeal swab and sputum specimens can be used with the XABTSARS-CoV-2 kit.

Human oropharyngeal swab, nasopharyngeal swab and sputum specimens may be used with the XABTSARS-CoV-2 kit. The swab sampling head should be cotton and/or medical absorbent cotton. Swabs should

be satisfied with the standards of the local medical device regulations. The transport media should besterile normal saline, tissue culture medium (DMEM) or pH7.0 phosphate buffer (Hank's solution, PBS).

7.1.1 Oropharyngeal swab and nasopharyngeal swab specimens are collected according to the TechnicalGuidelines for Covid-19 Laboratory Testing (Fifth Edition).

After verification, the following preservation solutions can be used: 1 mL sterile normal saline, tissue

culture medium (DMEM) or pH7.0 phosphate buffer (Hank's solution, PBS).

After verification, swab sampling head can use cotton swab and medical absorbent cotton.

Requirements of sampling tube: external screw mouth, resistant to - 80 ℃ freezing. Or sterile test tube.

Sterile cotton ball or other sealed covers can be used to tightly plug the test tube for airtight inspection.

7.1.2 Sputum samples are collected according to the Technical Guidelines for Covid-19 Laboratory Testing

(Fifth Edition). The volume of collected sputum sample shall not be less than 1mL.

After verification, sputum specimen can be added into an international general-purpose spiral coversputum bottle, a sterile sputum collector, or 50mL test tube with a spiral cover. Depending on the viscosity

of the sputum, add 1-4 times volume of 4% NaOH digestive fluid into the sputum tube, and tighten thespiral cover, vortex oscillate for 1 minute. And place it in the biosafety cabinet at room temperature for 15-

20 minutes, until the sputum is fully liquefied. Or add the same volume of acetylcysteine (10g / L) into thesputum sample, shake at room temperature for 30 minutes, and then carry out the subsequent nucleic acid

extraction after full liquefaction.

7.2 Specimen Storage

Keep refrigerated (2oC ~ 8oC) if it is to be processed within 24 hours.

Keep specimen frozen (-80oC) if it is to be processed after the first 24 hours or for extended periods within

one year (if there is no -80oC storage condition, they shall be temporarily stored in -20oC±5oC for no morethan 2 weeks).



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7.3 Shipment

Ship within 48 hours. Ship (insofar as possible) with dry ice; at the very least, maintain the cold chain withcooling gels. Always use triple packaging.

If shipment is delayed and facilities are available, the specimens should be frozen at -80oC and shipped ondry ice.

8. RNA Extraction Procedure for Specimens and Controls

The nucleic acid extraction reagents are not supplied in this kit and four commercial kits are recommended

for SARS-CoV-2 RNA extraction:

1) Nucleic acid extraction Kit (XABT, Cat. # CN8033);

2) QIAamp Viral RNA Mini Kit (Qiagen, Cat. # 52904 or 52906);

3) PURELINK VIRAL RNA/DNA KIT (Invitrogen, Cat. #12280050);

4) High Pure Viral RNA Kit (Roche, Cat. # 11858882001).

It is noted that the positive control and negative control in this kit should be extracted with the same

protocol for specimens. The internal control in this kit should be added into the extraction mixture with 10μL/test to monitor the whole process.

8.1 Test Preparation

8.1.1 Instruments and Materials Preparation

Instruments and materials required for extraction but not provided are listed below:

Freezer (-20oC±5oC, -80oC) and refrigerator (2oC ~ 8oC)

Vortex

Microcentrifuge† (with rotor for 1.5mL and 2mL tubes)

RNase-free microtubes

Heating block or water bath for lysis of specimens at 56 oC

Tube racks

Class II biosafety cabinet (or glove box)

Personal protective equipment: Powder-free gloves, lab coat, eye protection, etc.

Pipets† and pipette tips (To prevent cross-contamination, we strongly recommend the use of pipette



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tips with aerosol barriers)

Ethanol (96 ~ 100%)*

Nucleic acid extraction Kit (XABT, Cat. # CN8033) or QIAamp Viral RNA Mini Kit (Qiagen, Cat. # 52904 or

52906) or PURELINK VIRAL RNA/DNA KIT (Invitrogen, Cat. #12280050) or High Pure Viral RNA Kit(Roche, Cat. # 11858882001).

Notes:

* Do not use denatured alcohol, which contains other substances such as methanol or Methylethylketone.

† To ensure that specimens are properly processed according to the Kit procedures, we strongly recommend

that instruments (e.g., pipettes and heating blocks) are calibrated according to the manufacturers’recommendations.

8.1.2 Specimens Preparation

Appropriate specimens are oropharyngeal swab, nasopharyngeal swab or sputum. The specimens can bestored at 2oC ~ 8oC for up to 24 hours. For long-term storage, freezing at –20oC±5oC or -80oC in aliquots isrecommended (refer to Section 7.2). Frozen specimens must not be thawed more than once. Repeated

freeze–thawing leads to denaturation and precipitation of proteins, results in reduced viral titers and

therefore reduces yields of viral nucleic acids. In addition, cryoprecipitates might be formed during freeze–thawing. If cryoprecipitates are visible, they can be pelleted by centrifugation at approximately 6,800 x g for3 min. The cleared supernatant should be removed and processed immediately without disturbing the

pellet.

8.2 RNA Isolation

8.2.1 XABT-Nucleic acid extraction Kit

Note:

a. Different batches of different components in different batch kits are not interchangeable.

b. In Washing Buffer and Rinsing Buffer to be add anhydrous ethanol before the first use. Please prepareanhydrous ethanol (analytical purity). The details see instruction.

c. Carrier RNA is in the form of transparent jelly. When it is used, 300 μL eluent is added, and vortexoscillation makes it completely dissolved.

1) After adding 10 μL protease K and 6 μL carrier RNA into 1.5mL sterile centrifuge tube, followed by

adding 200μL specimen, or positive control or negative control and 10μL internal control and 200 μL



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Lysis Buffer into the centrifuge tube, mix them with vortex shaking for 15 seconds, centrifuge them5000rpm for 5 seconds, and place them at room temperature for 5 minutes.

Note: Specimen less than 200uL should be supplemented with PBS or normal saline.

2) Add 400 μL anhydrous ethanol and mix for 15 seconds by vortex oscillation with the tube covered.Centrifuge immediately for 5000rpm for 5 seconds after thorough mixing, and place at room

temperature for 5 minutes.

3) Transfer all the mixed liquid to the spin column (put the spin column in the collection tube). With

covering the tube, Centrifuge at 12,000 rpm for 1 minute, and put the spin column into the newcollection tube.

4) Add 500 μL Washing Buffer to the spin column, centrifugate at 12,000 rpm for 30 seconds aftercovering the tube, and put the spin column into the new collection tube.

5) Add 500 μL Rinsing Buffer to the centrifuge column, cover the tube and centrifuge at 12000 rpm for 30seconds. Place the centrifuge column in the new collection tube.

6) After centrifuging at 12000 rpm for 3 minutes to remove the residual Rinsing Buffer, put the centrifuge

column into a new RNase-Free centrifuge tube (1.5mL), followed by carefully opening the cover of thecentrifuge column, and drying it at room temperature for 3 minutes.

7) Add 100 μL eluent to the center of the spin column, and place it at room temperature for 3 minutes

with the tube covered. Centrifugation at 12000 rpm for 1 minute to collect eluent.

8) The virus nucleic acid obtained after centrifugation can be used for PCR detection or stored at -70 oC ~

-90 oC.

8.2.2 QIAGEN-QIAamp Virus RNA Mini Kit

Note: QIAamp Mini spin columns should be stored dry at room temperature (15oC~25oC); storage at highertemperatures should be avoided. All solutions should be stored at room temperature unless otherwise

stated. QIAamp Mini spin columns and all buffers and reagents can be stored under these conditions untilthe expiration date on the kit box without showing any reduction in performance.

Lyophilized carrier RNA can be stored at room temperature until the expiration date on the kit box. CarrierRNA should be dissolved in Buffer AVE; dissolved carrier RNA should be immediately added to Buffer AVL as

described on page 14. This solution should be prepared fresh, and is stable at 2oC~8oC for up to 48 hours.Buffer AVL–carrier RNA develops a precipitate when stored at 2oC~8oC that must be redissolved by warming

at 80oC before use. Unused portions of carrier RNA dissolved in Buffer AVE should be frozen in aliquots at –20oC. Do NOT freeze–thaw the aliquots of carrier RNA more than 3 times.

DO NOT warm Buffer AVL–carrier RNA solution more than 6 times. DO NOT incubate at 80oC for more than



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5 min. Frequent warming and extended incubation will cause degradation of the carrier RNA, leading toreduced recovery of viral RNA and eventually false negative RT-PCR results, particularly when low-titerspecimens are used.

1) Pipette 560µL of prepared Buffer AVL containing carrier RNA into a 1.5mL microcentrifuge tube.

2) Add 140µL of specimen, or positive control or negative control and 10µL internal control to the Buffer

AVL-carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing for 15 sec.

3) Incubate at room temperature (15oC~25oC) for 10 min.

4) Briefly centrifuge the tubes to remove drops from the inside of the lid.

5) Add 560µl of absolute ethanol (96%-100%) to each tube, and mix by pulse-vortexing for 15 sec. After

mixing, briefly centrifuge the tubes to remove drops from inside the lid.

6) For each specimen and control, place a QIAamp spin column into a 2mL collection tube (from the

QIAamp Viral RNA Mini Kit). Be sure to label the top of the columns clearly.

7) Carefully transfer the mixture from Step 5, including any precipitate, to the QIAamp spin columnWITHOUT wetting the rim of the column.

8) Centrifuge 1-2 min at 6,000 x g. If the specimen has not cleared the filter after the first run, repeatcentrifugation until the specimen has cleared the filter.

9) For each specimen and control, place the QIAamp spin column into a clean 2mL collection tube (from

the QIAamp Mini Kit) and add 500µl of Buffer AW1. Discard the tube containing the filtrate from theprevious step.

10) Centrifuge 1-2 min at 6,000 x g. If the buffer has not cleared the filter after 1-2 min, repeat

centrifugation until buffer has cleared the filter.

11) Place each QIAamp spin column into a third clean 2mL collection tube (from the QIAamp Mini Kit).

Carefully open the QIAamp spin column and add 500μL of Buffer AW2.

12) Centrifuge at full speed (approx. 14,000 x g) for 3 min. Discard the tube containing the filtrate from theprevious step.

13) To eliminate any possible Buffer AW2 carryover, place the QIAamp spin column into a new collection

tube, discard the old collection tube, and centrifuge at full speed (approx. 14,000 x g) for 1 min.

14) Place the QIAamp Mini column in a clean, clearly labeled 1.5mL RNase-free microcentrifuge tube (not

provided). Discard the old collection tube containing the filtrate.

15) Carefully open the QIAamp Mini column and add 60μL of Buffer AVE that has been equilibrated toroom temperature. Close the cap, and incubate at room temperature for 1 min.



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16) Centrifuge at 6,000 x g for 1 min. RNA is now present in the eluate and ready to test. Store specimensand controls at 2oC~8oC until PCR master mixes are prepared.

17) Extracted specimens should be tested by PCR within 6 hours of completing the extraction process.

Redundant specimens should be stored at 2oC~8oC while testing is in progress. Long-term storage ofextracted specimens (>6 hours) should be at -20oC (preferably -80oC). Minimize (do not exceed 3)

repeated freeze-thaw cycles.

Note: For more information, please refer to the manufacturer’s instructions.



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Figure 1. Viral RNA Isolation Procedure using QIAamp Viral RNA Mini Kit. The illustration

shows the simple steps (lyse, bind, wash, and elute) of the spin protocol and the vacuumprotocol.



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Figure 2. Viral RNA Isolation Procedure using XABT RNA Isolation Kit(Paramagnetic Beads Column). The illustration shows the simple steps (lyse, bind,

wash, and elute) of the protocol.

8.2.3 Invitrogen-PURELINK VIRAL RNA/DNA KIT

Note: The recommended purification protocol uses 5.6 μg Carrier RNA per specimen (for ≤500 μLspecimen). If you wish to use less Carrier RNA per specimen, you need to validate the amount of



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Carrier RNA needed for each specimen type and downstream application.

Add 60 mL 96–100% ethanol to 15 mL Wash Buffer (WII)included with the kit.Place a check in the boxon the Wash Buffer label to indicate the ethanol is added. Store the Wash Buffer (WII) with ethanolat room temperature.

1) Add 25 μL Proteinase K (included with the kit) into asterile microcentrifuge tube.Add 200 μL ofspecimen, or positive control or negative control and 10μL internal control into the

microcentrifuge tube.

2) Add 200 μL Lysis Buffer (containing 5.6 μg Carrier RNA).Close the tube lid and mix by vortexing

for 15 seconds.Incubate at 56oC for 15 minutes.Briefly centrifuge the tube to remove any dropsfrom the inside of the lid.

3) Add 250 μL 96–100% ethanol to the lysate tube to obtain a final ethanol concentration of 37%,close the lid, and mix by vortexing for 15 seconds.

Note: If you are processing up to 10 specimens, you may add ethanol to all tubes and thenvortex each tube.

4) Incubate the lysate with ethanol for 5 minutes at room temperature.Briefly centrifuge the tube

to remove any drops from the inside of the lid.

5) Transfer the above lysate with ethanol (~675 μL) onto the Viral Spin Column. Close the lid and

centrifuge the column at ~6800 × g for 1 minute. Discard the collection tube with the

flowthrough.

Note: If you are processing >200 μL starting material, you need to perform multiple loadings ofthe lysate by transferring any remaining lysate to the same Viral Spin Column and centrifuge at

6800 × g for 1 minute.

6) Place the spin column in a clean Wash Tube (2 mL) included with the kit and add 500 μL Wash

Buffer (WII)with ethanol to the spin column. Close the lid and centrifuge the column at ~6800 ×g for 1 minute. Discard the flow-through and place the spin column back into the Wash Tube.

Note: Additional Wash Tubes are available separately, if you do not wish to reuse the WashTube.

7) Add 500 μL Wash Buffer (WII) with ethanol into the spin column. Close the lid, centrifuge at~6800 × g for 1 minute. Discard the Wash Tube containing the flow-through.

8) Place the spin column in another clean, Wash Tube (2 mL) included with the kit. Centrifuge thecolumn at maximum speed in a microcentrifuge for 1 minute to dry the membrane completely.Discard the Wash Tube with the flowthrough.



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9) Place the Viral Spin Column in a clean 1.5-mL Recovery Tube supplied with the kit.Add 10–50 μL

of Sterile, RNase-free water to the center of the column. Close the lid.

Note: You may use an elution volume of ≤150 μL for elution. Incubate at room temperature for 1minute. Centrifuge the column at maximum speed for 1 minute. The Recovery Tube contains

purified viral nucleic acids. Remove and discard the spin column. Store the purified RNA/DNA at

–80oC or use the RNA/DNA for the desired downstream application.

Figure 3. The flow chart for purifying viral RNA/DNA using the PureLink™ Viral RNA/DNAMini

Kit is shown above.



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8.2.4 Roche-High Pure Viral RNA Kit

Note: Poly (A) carrier(Vial 2)Dissolve poly(A) carrier RNA (vial 2) in 0.4 mL Elution Buffer (vial 4).

Prepare aliquots of 50 μL for running 8 × 12 purifications. Prepare aliquots of 100 μL for running 4 ×25 purifications. For 12 purifications, thaw one vial with 50 μL poly(A) carrier RNA and mixthoroughly

with 5 mL Binding Buffer (vial 1). For 25 purifications, thaw one vial of 100 μL poly(A) carrier RNA andmix thoroughly with 10 mL Binding Buffer (vial 1).

Add 20 mL absolute ethanol to Inhibitor Removal Buffer. Label and date bottle accordingly after

adding ethanol.

Add 40 mL absolute ethanol to Wash Buffer. Label and date bottle accordingly after adding ethanol.

1) To a nuclease free 1.5 mL microcentrifuge tube: Add 200 μL specimen, or positive control ornegative control and 10 μL internal contro.Add 400 μL Working solution [Carrier RNA

supplemented Binding Buffer] and mix well.The RNA yield can be increased twofold by anoptional incubation step, thus resulting in higher sensitivity.After adding the Binding Buffer to

the specimen, simply incubate the mixture at +15oC to +25oC for 10 min. Thisincubation step canbe omitted when time to result is critical.

2) To transfer the specimen to a High Pure Filter Tube: Insert one High Pure Filter Tube in oneCollection Tube. Pipette entire specimen into the upper reservoir of the Filter Tube.Insert the

entire High Pure Filter Tube assembly into a standard tabletop centrifuge.Centrifuge the tubeassembly 15 sec at 8,000 × g. After centrifugation: Remove the Filter Tube from the Collection

Tube, discard the flowthrough liquid, and the Collection Tube.Insert the Filter Tube into a newCollection Tube.

3) After re-inserting the Filter Tube:Add 500 μL Inhibitor Removal Buffer to the upper reservoir of

the Filter Tube assembly and centrifuge 1 min at 8,000 × g. Discard flowthrough and combineFilter Tube with a new Collection Tube.

4) After removal of inhibitors:Add 450 μL Wash Buffer to the upper reservoir of the Filter

Tube.Centrifuge 1 min at 8,000 × g and discard the flowthrough.

5) After the first wash and centrifugation:Remove the Filter Tube from the Collection Tube, discardthe flowthrough liquid, and the Collection Tube.Insert the Filter Tube into a new Collection

Tube.Add 450 μL Wash Buffer to the upper reservoir of the Filter Tube. Centrifuge 1 min at8,000 × g.

6) Leave the Filter Tube-Collection Tube assembly in the centrifuge and spin it for 10 sec atmaximum speed(approximately 13,000 × g) to remove any residual Wash Buffer.The extra



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centrifugation time ensures removal of residual Wash Buffer.Discard the Collection Tube and

insert the Filter Tube into a clean, sterile 1.5 mL microcentrifuge tube.

7) To elute the viral RNA: Add 50 μL Elution Buffer to the upper reservoir of the Filter Tube.Centrifuge the tube assembly for 1 min at 8,000 × g.The microcentrifuge tube now contains the

eluted viral RNA. Either use the eluted RNA directly in RT-PCR or store the eluted RNA at −80oCfor later analysis.

Figure 4. Experimental overview

8.3 Test Method

When the kit is used for the first time, it is taken out from the -20 ℃ refrigerator and all the

components can be taken out in advance, melted at room temperature, briefly shaken, andinstantaneously centrifuged. Remove the kit from the refrigerator when it is used again, shake briefly,

and centrifuge instantly.

8.3.1 Sample handling (Sample handling area)

Add 10μL of internal control into 200μL of sample, positive control and negative control, respectively.It is recommended to use nucleic acid extraction reagent (Beijing Changping Medical Device Filing NO.

20200008) of Beijing Applied Biological Technologies Co., Ltd. for nucleic acid extraction.

200 μL specimen



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8.3.2 Reagents preparation (reagent preparation area):

1) Reaction system preparation: Take out the reagent and thaw the reagent completely. Invert themixture and centrifuge immediately. N test reactions (N = number of samples to be tested (n) +

positive control (1) + negative control (1) + 1) are prepared for reaction systems, respectively, asfollows.

Components Reaction system A Reaction system B

Nuclease-free water 2µL×N 2µL×N

Nucleic acid amplification reactionsolution

10µL×N 10µL×N

20×Reverse transcriptase 1µL×N 1µL×N

10×reaction solution (A/B) 2µL×N 2µL×N

Total volume 15µL×N 15µL×N

2) Reaction system distribution: Mix and centrifuge the above reaction solution, and dispense an

amount of 15μL each tube in a PCR tube suitable for a fluorescence PCR instrument.

8.3.3 Loading (Sample handling area)

5μL of the extracted sample nucleic acid, positive control and negative control are added to the Aand B reaction systems, and the total reaction volume is 20μL. Don’t mix, fasten the PCR tube cover,

the liquid on the tube wall was thrown to the bottom of the tube by instantaneous centrifugation,and then perform the PCR amplification immediately

After verification, add addition 5μL-10μL paraffin oil in the PCR system can prevent contamination

and avoid evaporation with no effect on the results



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Instrument Specific Instructions

9. Set Up

9.1 Operation Procedure on ABI 7500

1) Double click on the ABI system icon on the desktop to start the program.

2) Select “Advanced Setup” from the Quick start up menu. The “ExperimentProperties”screen will appear. Change the Experiment name.

3) Click “Plate Setup” and a new screen will appear as below.

4) If there is no “VIC” and “CY5”, click “Add New Target” .

5) Select the Reporter (FAM、 VIC & CY5) and change “Quencher” to “None”.



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6) Click “Assign Targets and Specimens”, select the well containing the specimens and controls,

and then click the Assign. Change “ROX” to “None”.

7) Click “Run Method”, Set the parameters as follows:

8) Save the document and then click “Run” to start the evaluation.



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9) PCR Analysis: After the run is completed, click “Analysis”. Click “Target” to select a fluorescent

channel, and view and adjust the “Threshold”. Lastly, be sure to click “Reanalyse” to update the

analysis.

10) Click “Export” icon above the graph to display the cycle threshold (Ct) values.

9.2 Operation Procedure on Bio-Rad CFX 96

1) 9.2.1 Double-click the icon to open the Bio-Rad CFX software.

2) 9.2.2 Create a new Experiment and choose the type of the instrument, click “OK”.

3) Under “Protocol” interface, click the “Create New” button to create a new protocol or click the“Select Existing” button to invoke the existed protocol file.



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After clicking “Create New” button, you can see the interface below. Insert a stepfirst.

4) Parameter setting:

Step 1 45oC for 10 min1 cycle

Step 2 95oC for 5 min

Step 3 95oC for 15 sec

45 cyclesStep 4

60oC for 45 sec

*Fluorescence measured at 60oC

Specimen volume: 20 μL



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5) Click “OK”. Save the protocol.

6) Under “Plate” interface, click “Create New” button to create a new plate or click “Select Existing”

button to invoke the existed plate file.

7) Select the fluorescence channel: Click “ ” to choose “FAM”,“VIC” and “Cy5” channel, click “OK”.



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8) Choose the well and select specimen type in the drop-down list box. Choose “Unknown” forspecimen, “Positive Control” for positive control and “Negative Control” for negative control.

9) Choose all the wells in the experiment. For Tube A click “FAM", “VIC" and “Cy5"; for Tube Bclick “FAM", “VIC" and “Cy5".



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10) Click “OK”. Save the plate.

11) Under “Start Run” interface, click “Open Lid”. Add the plate into the instrument and then click

“Close Lid”. Click “Start Run”, and then save the run file. The experiment begins.



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12) PCR Analysis: After the run is completed, click the well to read the Ct value on the lower right

and amplification curve on the upper left.

9.3 Operation Procedure on Roche Light Cycler480Ⅱ/Roche Cobas z 480

1) Double-click the icon to open the LightCycler480 software.

2) Enter the correct user name and password in the software login interface, and click

3) In the software operation interface, click ， Create a new Experiment.



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4) Reaction program settings

Selection of fluorescent channel: click “Customize” in the operation interface, select the

corresponding fluorescent channel(FAM,VIC,CY5), and Click .



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Reaction procedure and step setting: Set reaction volume (20 μL) in “Reaction volume”, Click ，set

up reaction steps，In“Cycle” and “Analysis mode”, set Cycle and Analysis Mode (Quantification), and

select “single” in “Acquisition Mode” . The specific program settings are as follows:

Step 1 45oC for 10 min1 cycle

Step 2 95oC for 5 min

Step 3

95oC for 15 sec

45 cycles60oC for 45 sec

*Fluorescence measured at 60oC



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5) Specimen name setting

click “Specimen Editor” to name the specimen in“Specimen Name”,Click 。

6) Operation procedure

Click “Start Run” in the lower right corner ,Save the file , theprogram begins.

7) Result analysis

Click “Analysis” to analyze the results, select “AbS Quant / Fit Points”, Click , in the resultanalysis interface, Click “Calculate” to analyze and calculate, then the fluorescence curve and Ctvalue can be obtained in “AbS Quant result”



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Interpretation of Results

10. Interpretation of Controls and Clinical Specimens

10.1 Controls

Test Run is Valid when All Controls Meet the Following Stated Standards.

Negative control reaction must be negative without an amplification curve at FAM and VIC channelsand Ct value≤38 at Cy5 channel. If failed, the test run is invalid and potential sources ofcontamination should be identified and corrected (see Table 1).

Positive control reaction should be positive at FAM and VIC channels, with Ct value ≤38. If failed, it isrecommended to re-test the run using new reagents (see Table 1).

Table 1: Interpretation of Controls

Detection channels Negative control Positive control

Tube A

FAM(ORF1ab) No Ct Ct≤38

VIC(N) No Ct Ct≤38

Cy5(IC) Ct≤38 ——

Tube BFAM(E) No Ct Ct≤38

Cy5(IC) Ct≤38 ——

10.2 Specimen

1) When all controls are performed correctly, the run is valid.

2) The amplification curve of internal control(CY5) shows a typical S curve and Ct≤38, the

interpretation of the results for target genes is subjected to the following conditions (see Table

2).

Table 2: Interpretation of target genesDetection channels

Interpretation the results of target genesFAM VIC

Tube ACt≤38 Ct≤38

With a typical S amplification curve, ORF1ab gene(FAM)and/or N gene(VIC) is positive.

38＜Ct＜40 38＜Ct＜40With a typical S amplification curve, re-test thecorresponding target gene of the specimen again. If the Ct



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value＜40 with a typical S amplification curve, thecorresponding target gene is positive; if the Ct value≥40, thecorresponding target gene is negative.

Ct≥40 Ct≥40 The corresponding target gene is negative.

Tube B

Ct≤38 ---With a typical S amplification curve, E gene (FAM) ispositive.

38＜Ct＜40 ---

With a typical S amplification curve, re-test the target gene ofthe specimen again. If the Ct value＜40 with a typical Samplification curve, the E gene is positive; if the Ct value≥40,the E gene is negative.

Ct≥40 --- The E gene is negative.

3) According to the results of ORF1ab and N gene in tube A, and the result of E gene in tube B,interpretation as follows: If TWO or THREE genes of the detected genes are positive, the SARS-

CoV-2 is positive; If ONLY ONE or NONE of the detected genes is positive, the SARS-CoV-2 isnegative.

4) The amplification curve of the positive specimen should be with a typical S curve. However, ifthe target concentration is too high, the internal standard control may not be amplified and thespecimen can be directly judged as positive. If any two of target genes get Ct≤38, the SARS-CoV-

2 is positive. If any two of target genes get Ct≥40, the SARS-CoV-2 is negative. If Ct≥40, or show

no value, interpretation the results of target gene is negative.

5) If all of the Ct values of FAM, VIC and Cy5 channels are more than 38 or there is no obvioustypical S amplification curve, suggestions are as follows:

• There is/are substance(s) in the specimen that inhibit the PCR reaction. It is recommendedto dilute the specimen for 5-10 times to be re-tested.

• The process of nucleic acid extraction is abnormal, so it is suggested to re-extract nucleic

acid for re-test.

• This specimen is not a qualified specimen at the time of sampling, or has degraded duringtransportation and storage.



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11. Limitations

• The test results of this kit are for clinical reference only. Comprehensive considerationshould be given to the clinical diagnosis and treatment of patients in combination with

their symptoms, medical history, other laboratory tests, and treatment response.

• The target sequences detected by this kit are conserved region of the SARS-CoV-2 genes.

Mutations in the target sequences or sequence changes due to other causes may result infalse negative results.

• If E gene is positive, both ORF1ab and N genes are negative, the SARS-CoV-2 is negative.Further inspections are recommended.

• For emergent respiratory viruses, the optimal specimen type for detection and the optimal

sampling time after infection may not be confirmed. Therefore, the possibility of false

negative results can be reduced by multiple sampling in the same patient.

• Unreasonable sampling, delivery and storage conditions, and low virus concentration (＜

200 copies/mL) in the specimen may lead to false negative results.

• Unverified other interference or PCR inhibitors may result in false negative results.

• If the cross-contamination is not controlled during specimen processing, and if otherpathogens containing the target nucleic acid sequence may lead to false positive results.

• The correlation between copies /mL and PFU/mL is not confirmed.



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12. Analytical Performance Characteristics

12.1 Limit of Detection

SARS-CoV-2 simulation specimens were examined in the analytical sensitivity tests. The specimenswere diluted linearly into various concentrations. The tests were performed with three lots of kits.

Each test was repeated for 3 times. Then confirmatory tests for the positive specimens with lowestSARS-CoV-2 concentration were further repeated for 20 times. The results suggested that the

minimum detection for SARS-CoV-2 is 200 copies/mL.

12.2 Analytical Specificity

a) Interfering substances

There is no significant effect on the test results for verification of 5% blood, mucin (≤0.9mg/mL), and

external or internal use of common drugs for patients with colds and other respiratory symptomssuch as Benorin, hydroxymezoline, sodium chloride (including preservatives), beclomethasone,

dexamethasone, flunisolone, triamcinolone acetonide, budesonide, mometasone, fluticasone,

histamine hydrochloride, interferon α, zanamivir, ribavirin, oseltamivir, palamivir, lopinavir, ritonavir,abidol, levofloxacin, azithromycin, ceftriaxone, meropener Penem, tobramycin were verified, therewas no significant impact on the results.

b) Cross reactivity

Related microorganisms and human genomic DNA specimens were examined by wet-testing and/oranalyzed in silico, some of microorganisms and human genomic DNA were tested with XABT SARS-

CoV-2 in the specificity tests. The tests were performed with three lots of kits (Lot# 20200101,20200102, 20200103). All tests were performed according to the User Multiple Real-Time PCR Kit for

Detection of 2019-nCoV. The tests were conducted in February 2020. Our results showed that the kitonly detected SARS-CoV-2; whereas negative SARS-CoV-2 detection was observed using other 56microorganisms and human genomic DNA specimens, suggesting high specificity of the kit for SARS-

CoV-2 detection. The test results with 56 microorganisms and human genomic DNA specimens are

summarized in the following table. The other 14 microorganisms, including Influenza C, Parechovirus,Candida albicans, Corynebacterium diphtheriae, Legionellanon-pneumophila,Bacillus anthracosis (Anthrax), Moraxella cararrhalis, Neisseria elongate and miningitidis,

Pseudomonas aeruginosa, Staphylococcus epidermis, Staphylococcus salivarius, Leptospirosis,Chlamydia psittaci, Coxiella burneti (Q-Fever) , Streptococcus aureus, had no cross-reference with

SARS-CoV-2 by in silico.



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Table: Specificity Test ResultsSpecimen Lot #

20200101 20200102 20200103SARS-CoV-2

InternalControl

SARS-CoV-2

InternalControl

SARS-CoV-2

InternalControl

Human coronavirus 229E - + - + - +Human coronavirus OC43 - + - + - +Human coronavirus hku1 - + - + - +Human coronavirus nl63 - + - + - +

MERS-coronavirus - + - + - +SARS-coronavirus - + - + - +

Influenza A - + - + - +Influenza B Yamagata - + - + - +Influenza B Victoria - + - + - +

Respiratory syncytial virus A - + - + - +Respiratory syncytial virus B - + - + - +

Rhinovirus A - + - + - +Rhinovirus B - + - + - +Rhinovirus C - + - + - +

Parainfluenza virus 1 - + - + - +Parainfluenza virus 2 - + - + - +Parainfluenza virus 3 - + - + - +Parainfluenza virus 4 - + - + - +

Adenovirus 1 - + - + - +Adenovirus 2 - + - + - +Adenovirus 3 - + - + - +Adenovirus 4 - + - + - +Adenovirus 5 - + - + - +Adenovirus 7 - + - + - +Adenovirus 55 - + - + - +

Enterovirus Type A - + - + - +Enterovirus Type B - + - + - +Enterovirus Type C - + - + - +Enterovirus Type D - + - + - +

Human Metapneumovirus (hMPV)

- + - + - +

EB virus - + - + - +Measles virus - + - + - +

Human cytomegalovirus - + - + - +Rotavirus - + - + - +



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Norovirus - + - + - +Mumps virus - + - + - +

Varicella zoster virus - + - + - +Mycoplasma pneumoniae - + - + - +Chlamydia pneumoniae - + - + - +Legionella pneumophila - + - + - +Bordetella pertussis - + - + - +

Haemophilus influenzae - + - + - +Staphylococcus aureus - + - + - +

Streptococcus pneumoniae - + - + - +Streptococcus pyogenes - + - + - +Klebsiella pneumoniae - + - + - +

Mycobacterium tuberculosis - + - + - +Aspergillus fumigatus - + - + - +

Smooth Candida - + - + - +Cryptococcus neoplasia - + - + - +

Pneumocystis jirovecii (PJP) - + - + - +Human Genome DNA - + - + - +

12.3 Precision

Precision covers repeatability and reproducibility (i.e. inter-lot, inter-day, inter-run, inter-site, inter-

operator and inter-instrument), for which 3 lots of assays and 3 different PCR instruments in totalwere employed for the study.

Precision study was performed with 3 lots of assays on 3 different PCR instruments (ABI Prism®7500,

Bio-Rad CFX96 and Roche LC480 II). We tested enterprise precision reference which contains 2different concentrations (J1: 5E+03 copies/mL, J2: 1E+03 copies/mL) and clinical specimens which

contains 3 different concentrations oropharyngeal swab specimens (S3: 5E+03 copies/mL, S1: 1E+03

copies/mL ,SN-Y: 0), nasopharyngeal swab specimens (S4: 5E+03 copies/mL, S6: 1E+03copies/mL ,SN-Y: 0) and 3 different concentrations sputum specimens (S5: 5E+03 copies/mL, S2:1E+03 copies/mL, SN-T: 0), with 10 replicates in a run for each concentration. Each day, two people,

at two different sites, each on three different platforms, repeated the experiment for 20 days. TheCV of all factors that affect it was computed with Ct values. The results are shown in Tables 4.

It shows that the ORF1ab, N, E gene CV among 3 lots, 3 instruments, 2 operators, 2 sites, 20 days are2.39%, 2.97%, 2.38% with J1; 3.04%, 2.98%, 2.87% with J2; 1.91%, 3.27%, 2.14% with S1; 3.32%,

2.62%, 1.87% with S2; 3.21%, 2.71%, 2.02% with S3; 3.64%, 2.72%, 2.09% with S4. And SN-T, SN-Yare always negative.



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Table 4: Diurnal differences of enterprise precision reference and clinical specimens(all extraction reagents, all instrument platform )

Reference CV of Lot-to-Lot/ day-to-dayJ1（ORF1ab） 2.39%

J1（N） 2.97%J1(E) 2.38%

J2（ORF1ab） 3.04%J2（N） 2.98%J2(E) 2.87%

S1（ORF1ab） 1.91%S1（N） 3.27%S1(E) 2.14%

S2（ORF1ab） 3.32%S2（N） 2.62%S2(E) 1.87%

S3（ORF1ab） 3.21%S3（N） 2.71%S3（E) 2.02%

S4（ORF1ab） 3.64%S4（N） 2.72%S4(E) 2.09%

13. Clinical Evaluation

A. Clinical evaluation in XABT lab

90 clinical positive specimens, ZCF001-ZCF090, were extracted and purified in the hospitals, and were

detected with Multiple Real-Time PCR Kit for Detection of 2019-nCoV(XABT) and ddPCR in ourcompany’s lab.

90 clinical negative specimens, ZCF091-ZCF180, were extracted and purified, and detected with

Multiple Real-Time PCR Kit for Detection of 2019-nCoV(XABT) in our company’s lab.

According to the results of the experiments described above, 20 of the clinical specimens at a

concentration of 1x-2x LoD, and 10 of the clinical specimens for very each specimen type

(orophryarngeal swab, nasopharygneal swab and sputum) at a concentration of 5x LoD, can bedetected by qPCR (Multiple Real-Time PCR Kit for Detection of 2019-nCoV(XABT) and confirmed byddPCR. In this evaluation for positive specimens, 100% positive specimens can be detected. For the

negative specimens, 90 negative specimens(30 cases for each specimen types, orophryarngeal swab,nasopharygneal swab and sputum), and none of the specimens was detected as positive



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interpretation. And in the study for Analytical Specificity, 55 cases as negative specimens were

detected and none of them were detected as positive for very target and result interpretation.

In summary, in this section for clinical evaluation in our lab for 90 positive specimens and 90 negativespecimens, 100% agreement for positive and negative specimens.

B. Clinical Evaluation in Clinical Institutions

Clinical evaluation was performed at three clinical Institutions of China. Specimens were collectedfrom patients exhibiting symptoms of SARS-CoV-2 infection from February 14, 2020 to February 18,2020. A total of 577 clinical specimens were obtained from 516 patients (including 62 patients with

sputum and swab paired specimens). The specimen types collected were: 167 sputum, 395oropharyngeal swab and 15 nasopharyngeal swab specimens.

All specimens were tested with the Multiple Real-Time PCR Kit for Detection of 2019-nCoV produced

by Beijing Applied Biological Technologies Co., Ltd.( XABT SARS-CoV-2) as well as the SARS-CoV-2 Kit(National Device Registration Permission No. 20203400057, approved by NMPA) produced byShanghai ZJ Bio-Tech Co., Ltd., used as a benchmark assay.

Of the 577 specimens included in the comparison study 238 (41.2%) were considered as positive forSARS-CoV-2 RNA with XABT SARS-CoV-2.

The results are summarized in the following table:

Table 5 Overall performance with oropharyngeal swabs, nasopharyngeal swabs and sputumspecimens

Results from XABT SARS-CoV-2Results from SARS-CoV-2 kit approved by NMPA

Positive Negative Total

Positive 220 18 238

Negative 2 337 339

Total 222 355 577

Overall performance of the XABT SARS-CoV-2:

- Positive Percent Agreement: 99.1% (220/222) - 95% C.I. 96.8-99.9%

- Negative Percent Agreement: 94.9% (337/355) - 95% C.I. 92.1-97.0%



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References

1. Technical Guidelines for COVID-19 Laboratory Testing (Fifth Edition).

2. Prevention and control of novel coronavirus pneumonia (Fifth Edition)

3. General guidelines for biosafety in microbiology and biomedical laboratories(WS 233-2002) .

4. Medical waste management regulations.

5. Measures for the Management of Clinical Gene Amplification Testing Laboratories in

Medical Institutions.

WHO Emergency Use Assessment Coronavirus disease …...The Multiple Real-Time PCR Kit for Detection...

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