Renji Nucleic Acid Testing Kit Real Time RT-PCR Method ISO13485 Approval

Detection of SARS-CoV-2 in the Renji Coronavirus Nucleic Acid

based on amplification/detection of either the N or ORF1ab region

sequences. Therefore, the identified sequence variants in the 5′

end of the N gene forward primer are not predicted to affect

inclusivity for SARS-CoV-2.

【Product Name】

Novel Coronavirus (COVID-19) Nucleic Acid Detection Kit

(Real-time RT-PCR Method)

【Components of the Diagnostic Kit】

Note:

1. Do not mix or exchange components from different kit lots.

2. The COVID-19 Negative Control is sterile nuclease-free water, and the COVID-19

Positive Control is transcribed RNA invitro which contains target genes ORF1ab,

N gene, and Internal Control gene.

Renji Coronavirus Nucleic Acid Detection Kit is a real-time RT-PCR in vitro

diagnostic test intended for the qualitative detection of nucleic acid from

SARS-CoV-2 in human oropharyngeal swab and nasopharyngeal swab specimens

collected by a healthcare provider (HCP), and anterior nasal swab specimens

collected by an HCP or self-collected under the supervision of an HCP by any

individual, including individuals without symptoms or other reasons to suspect

COVID-19. This test is also for use with saliva samples collected using the Renji

Saliva Collection Kit either by an HCP or self-collected under the supervision of

an HCP in a healthcare setting from individuals suspected of COVID-19.

【Intended Use】

Renji Coronavirus Nucleic Acid Detection Kit is intended for use by qualified clinical

laboratory personnel specifically instructed and trained in the techniques of

real-time PCR and in vitro diagnostic procedures. The Coronavirus Nucleic Acid

Detection Kit is only for use under the Food and Drug Administration’s Emergency

Use Authorization.

2019-nCoV detection kit is used for in vitro qualitative detection of the ORF1ab and N
genes of SARS-CoV-2 RNA in nasopharyngeal swabs and sputum specimens of
suspected pneumonia cases, suspected cluster cases infected by novel coronavirus, and
other patients requiring diagnosis or differential diagnosis of the novel coronavirus
infection. The function of the assay is for the diagnosis or aid for diagnosis of COVID-19.
For the definitions of ‘suspected cases’ and ‘suspected clustered cases’, refer to the
documents (current version) such as ‘Diagnosis and Treatment Plan for Pneumonia
Infected by Novel Coronavirus’ and ‘Monitoring Plan for Pneumonia Cases Infected
by Novel Coronavirus’issued by CDC.

This product is only used in the pneumonia epidemic of SARS-CoV-2 infection since
December 2019. The auxiliary diagnosis of related cases and the in vitro diagnostic
epidemic reserve of this epidemic cannot be used as a conventional in vitro diagnostic
reagent for clinical application. In use, the relevant requirements of the ‘Pneumonitis
Diagnosis and Treatment Scheme for Novel Coronavirus Infection’ and ‘Pneumonitis
Prevention and Control Scheme for Novel Coronavirus Infection’should be complied.
To carry out nucleic acid detection of novel coronavirus, experimenters should conduct
biosafety procedures and conform to the requirements of ‘Technical Guidelines for
Laboratory Monitoring of novel coronavirus infected pneumonia’ by CDC.

This kit is used for the detection of COVID-19(ORF1ab/N Gene) Nucleic Acid and

assisted diagnosis and epidemiological surveillance of COVID-19.

【Features】

››› FAST: Complete results in 40-90 mins, from start to finish!

【Package Specification】

50 tests/kit & 200 tests/kit

【Test Principle】

The kit adopts the principle of real-time fluorescent quantitative PCR technology,

designs specific primers and probes for COVID-19(ORF1ab/N gene) ,

and combines them with real-time fluorescent quantitative PCR instrument to

detect the nucleic acid of COVID-19 virus. So as to realize the qualitative detection

of COVID-19 virus nucleic acid.

In addition, the PCR detection system uses the positive internal control,

which monitors the presence of PCR inhibitors in test specimens by detecting

whether the internal control signal is normal,to avoid a false negative result.

The assay is based on the real-time polymerase chain reaction (PCR) technology

and is composed of a ready to use optimized mixture with target specific primers

and probes for the detection of SARS-CoV-2. Probes consist of a fluorophore

covalently attached to the 5’-end of the oligonucleotide probe and a quencher at

the 3’-end. The quencher molecule quenches the fluorescence emitted by the

fluorophore when excited by the cycler’s light source. As long as the fluorophore

and the quencher are in proximity, quenching inhibits any fluorescence signals.

Degradation of the probe releases the fluorophore from it and breaks the proximity

to For Emergency Use Only Instructions For Use rev.1.2 2 the quencher, thus relieving

the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence

detected in the quantitative PCR thermal cycler is directly proportional to the

fluorophore released and the amount of DNA template present in the PCR.

【Applicable Instrument】

The diagnostic kit is applicable to MA-6000,ABI series, Bio-Rad series, Roche

LightCycler R480, Cepheid SmartCycler, Rotor-Gene series and other multi-channel

real-time quantitative PCR instruments.

【Specimen Requirements】

1. Sample types:Upper respiratory tract specimens(including throat swabs,

nasal swabs, nasopharyngeal extracts deep cough sputum), lower respiratory tract

specimens(including respiratory tract extracts, bronchial lavage fluid alveolar lavage

fluid, lung tissue biopsy specimens) , Tissue culture and other samples.

2.Storage conditions:The collected specimens should be submitted for inspection

in a timely manner, and the specimens should be stored at 4℃ within 24 hours.

It is best to store at-70℃ for more than 24 hours, and avoid repeated freeze-thaw

cycles.

【Storage and Stability】

1. The diagnostic kit should be stored in a sealed pouch at-20±5℃.The expiry date

is 12 months.

2. Please refer to the date of manufacture and expiry date on the outer package.

3. The reagent keep valid and stable within the expiry date if not used. The kit should

not be frozen and more than 5 times.

【Test Method】

1.Reagent Preparation (performed at “reagent preparation region”)

1.1 Takeout each component from the diagnostic kit and place them at room temperature.

Allow the reagents to equilibrate at room temperature, then vortex each of them

respectively for later use.

1.2 According to the quantity of test specimens, COVID-19 Positive Control and

COVID-19 Negative Control,pipette appropriate quantity of COVID-19 PCR Mix

and COVID-19 Enzyme Mix (COVID-19 PCR Mix 15 µl/test+COVID-19 Enzyme

Mix 5 µl /test) mix them thoroughly to make a PCR-Master mix centrifuge it

instantaneously for later use.

1.3 Transfer the above-prepared reagents to the "specimen processing region" for

later use.

2.Processing and loading of specimens (performed at "specimen

processing region")

2.1 This diagnostic kit does not include Viral RNA&DNA Extraction Kit. It is

recommended to use Viral RNA&DNA Extraction Kit produced by Changsha Renji

Medical Equipment Co, Ltd. to extract viral RNA. The specific operation is in

accordance with its instructions.

2.2 Add 20 µl PCR-Mastermix into PCR reaction tube with 5µl above processed sample,

COVID-19 Positive Control and COVID-19 Negative Control and cap the tube. Carry

out fluorescence quantitative PCR detection on fluorescence PCR instrument.

3.PCR Amplification (performed at "nucleic acid amplification area")

3.1 Place PCR reaction tubes into the specimen wells of the amplification equipment

Setup COVID-19 Positive Control COVID-19 Negative Control and specimens to be

tested in the corresponding sequence and input specimen name.

3.2 Set cycle parameters according to the following table for PCR amplification.

Note:

1) The fluorescence collection is set at "Step3:55°C, 30s". Selection of detection

channels:FAM, HEX and Cy5, where FAM channel is ORF1ab gene and HEX

channel is N gene. Cy5 channel is the internal control gene, and the reaction system

s set to 25 µl.

2) ABI series fluorescent PCR instruments do not select ROX calibration and select

None for the quenching group.

【Positive Judgement Value and Reference Interval】

1. Condition Setting for Result Analysis

The adjustment principle of Baseline and Threshold is generally based on the results

of the automatic analysis of the instrument. When the overall slope of the curve

appears, the Start, End,and Threshold values of the Baseline can be adjusted

according to the image. Usually, the user can adjust it according to the actual

situation. The Start value can be set to 3-15, and the End value can be set to 5-20.

Adjust the amplification curve of the negative control to make it straight or below

the thresh hold line.

2. The Validity of The Kit

2.1 The COVID-19 Positive Control:FAM, HEX and Cy 5 channels have typical

S-type amplification curves and Cts≤32.

2.2 The COVID-19 Negative Control:FAM, HEX and Cy 5 channels have no

Ct or Ct>38.

2.3 Note:The above conditions must be met at the same time, otherwise this

experiment is invalid and needs to be repeated.

3.The Positive Judgment Value

Through the study of reference values, it was determined that the Ct reference value

of the target gene and the internal control gene detected by this kit was both 38.

【QUALITY CONTROL】

The kit product provides negative control, positive control, and internal control to

monitor the reliability of the results for the entire batch of specimens from sample

extraction to PCR amplification. All test controls should be examined prior to

interpretation of patient results. Positive control, negative control and IC in positive

and negative control should meet the requirements listed in the below table to ensure

valid results. If the controls are not valid, the patient results cannot be interpreted.

Result Interpretation of Test Controls for 60 µL reaction:

Result Interpretation of Test Controls for 30 µL reaction:

Result Interpretation of Test Controls for 15 µL reaction:

1) Negative Control: both ORF1ab and N of SARS-CoV-2 must be not detected, and

the Ct value of internal control should be ≤ 40.

2) Positive Control: both ORF1ab and N of SARS-CoV-2 must be detected, and their

Ct values should fall within the ranges described in the above tables, the Ct value of

internal control does not have to be ≤40 for positive control.

【The Sample Result Judgment】

1.If the test sample detects atypical S-type amplification curve in the FAM, HEX

and Cy 5 channels and the Ct is≤38, the sample can be judged to be COVID-19

positive.

2.If the test sample has no Ct or Ct>38 in the FAM and HEX channels, and there

is atypical S-type amplification curve in the Internal Control channel(Cy 5) , Cts≤38,

the sample can be judged to be COVID-19 negative.

3.If the test sample only has a Ct values 38 in a single channel of the FAM or HEX

channel, and there is atypical S-type amplification curve in the Internal Control

channel(Cy 5) , Ct≤38, the results need to retest. If the results repeated are

consistent the sample can be judged as COVID-19 positive, the results repeated

are negative except for the typical S-type amplification curve of the Internal Control

channel(Cy 5) , Ct≤38, which could be judged as COVID-19 negative.

4.If no typical S-type amplification curve(No Ct value) or Ct value>38 is detected in

the FAM, HEX, and Cy 5 channels of the test sample, it means that there is a

problem with the quality of the sample or a problem with the operation. If the result

is invalid, you should find and eliminate the cause, collect the sample again,

and repeat the test(if the test result is still invalid, please contact the company) .

【Limitations of Detection Method】

1.Test results of the diagnostic kit can be used only for clinic reference. The clinical

diagnosis and treatment of patients should be considered in conjunction with their

symptoms, signs, medical history and other related conditions.

2.False negative results may occur when the concentration of the detected nucleic

acid in the test sample is below the minimum detection limit of this kit.

3.Improper handling of the tested sample during collection, transportation,

storage, and processing can easily result in RNA degradation and false negative

results.

4.When samples are cross-contaminated during collection, transportation, storage,

and processing, it is easy to get false positive results.

【Product Performance Index】

1.LOD:The limit of detection is 200 copies/ml.

2.Precision:Coefficient of variation (CV%] of precision Ct value within batch≤3%.

3.Specificity:There is no cross reaction between the kit and positive samples, such

as Human Coronavirus HCoV-NL63, Human Coronavirus HCoV-OC43, SARS

Coronavirus, MERS Coronavirus, Influenza A Virus, Influenza B Virus Yamagata,

Victoria, H1N1 Influenza Virus, H3N2 Influenza Virus, H5N1 Influenza Virus,

H7N9 Influenza Virus, Respiratory Syncytial Virus A, Adenovirus(type 2) ,

Adenovirus(type 2) , Mycoplasma Pneumoniae, Chlamydia Pneumoniae,

Pertussis, Streptococcus Pneumoniae, Rhinovirus(Type A) etc.

【Precautions】

1.The entire detection process should be performed strictly in accordance with the

requirements of this manual in the reagent preparation area, sample processing area,

and PCR amplification area, and the experimental clothes, instruments,

and consumables in each area should be used independently and cannot be mixed.

2.To avoid RNA degradation, the sample processing process should be operated at

0-4℃, and the test should be performed immediately after the experiment is

completed. Utensil consumables used in sample processing should be nuclease-free.

3.Negative and positive controls should be set for each experiment.

4.All reagents in the kit should be fully thawed and mixed at room temperature and

centrifuged immediately before use.

5.All negative and positive controls in the kit should be transferred to the sample

preparation area and stored separately before the first use.

6.To prevent fluorescence interference, avoid touching the PCR reaction tube

directly with bare hands, and avoid any marking on the PCR reaction tube.

7.The instrument amplification related parameters should be set in accordance with

the relevant requirements of this manual and different batches of reagents cannot

be mixed.

8.The product waste during the experiment should be detoxified before being

discarded.

【What is Nucleic Acid?】

【INDEX OF SYMBOL】

【EXPORTER】

Magnus International Limited

F12, New City International Mansion A, 234 Huapao Ave.

Liuyang, Hunan Province 410300 China

Contact: Goodwellmedical@gmail.com

【MANUFACTURER】

Changsha Renji Medical Equipment Co., Ltd.

No.18 Xiangtai Road, Liuyang Jingkai District,

Changsha City, Hunan Province 410300 China

【AUTHORIZED REPRESENTATIVE】

Lotus NL B.V.

Koningin Julianaplein 10, 1e Verd, 2595AA, The Hague, Netherlands.

【FACTORY VIEW】

【FACTORY WORKSHOP】

【PRODUCT AND STANDARDS】

【CERTIFICATIONS】

【FAQ】