Wenzhou Astock Biotechnogy Co.,Ltd

New Coronavirus COVID‐19 Nucleic Acid Detection Kit

(Fluorescence RT‐PCR Method) 

Instruction for Use 

PRODUCT NAME 

New Coronavirus COVID-19 Nucleic Acid Detection Kit( Fluorescent RT- PCR Method)

PACKAGING SPECIFICATIONS 

200 Test/Kit &50Test/Kit.

INTENDED USE 

This kit is used for the detection of COVID-19(ORFlabN Gene)Nucleic Acid and assisted diagnosis and epidemiological surveillance of COVID-19. 

TEST PRINCIPLE

This kit is based on the principle of fluorescent PCR technology, designed specific primers and Taqman probes for COVID-19(ORFlab/N gene), and detect them with a fluorescent PCR instrument to achieve detection of COVID- 19 nucleic acids.

In addition, the PCR detection system contains internal control primers and probes to monitor the specimen collection and the extraction process by detecting whether the internal standard is normal to avoid false negative results. 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 main source 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 main manifestations include fever, fatigue and dry cough. Nasal congestion, runny nose, sore throat, myalgia and diarrhea are found in a few cases.

MAIN COMPONENTS 

Number 25 Test/Kit 50Test/Kit 1 COVID-19 PCR Reaction Solution 425μLx1 tube 850μL×1tube 2 COVID-19 Enzyme Mixture 75μL x 1 tube 150μL x 1 tube 3 COVID-19 Negative Control 100μL x 1 tube 100μL x 1 tube 4 COVID-19 Positive Control 100μL x 1 tube 100μL x 1 tube 5 Instruction for use 1 Serving 1 Serving Note 1. Didderent batches of reagents cannot be mixed or interchanged. 2. The COVID-19 Negative Control is saline, and the COVID-19 Positive Control is transcribed RNA in vitro which contains target genes ORFlab, N gene, and Internal Control gene. 

STORAGE CONDITIONS AND EXPIRING DATA 

The kit should be stored at -20±5℃ and it is valid for 6 months. The production date and the expiration date can be seen in the outer packaging. The kit should not be frozen and thawed more than 5 times. Please use the reagent within 1 month after opening. 

APPLICABLE INSTRUMENT 

This kit is suitable for ABI series, Bio- Rad series, Agilent Stratagene MX series, Roche Lightcycler R480, Cepheid Smart Cycler, Rotor-Gene series and other multi-channel real-time quantitative PCR instruments.

SPECIMEN REQUIREMENTS

Specimen 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 tisssue biopsy specimens), Tissue culture and other specimens. 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 can be stored for more than 24 hours at -70℃, and avoid repeated freeze- thaw cycles.

TESTING METHOD

1.Reagent Preparation (reagent preparing area) Melt the components of the kit at 4℃, protected from light, and mix thoroughly before centrifugation.Calculate the number of reagents used N (N =number of specimens + 1 positive control+ 1 negative control), configure the reaction system mix according to the following table, add an appropriate volum of centrifuge tube, fully mix and centrifuge immediately, aliquot according to 20ul to a PCR reaction tube/plate and transfer to the specimen processing area. Component Volume（ul） COVID-19 PCR Reaction Solution 17 COVID-19 Enzyme Mixture 3 Total capacity (reaction system mix) 20 

2.Processing and Extraction of the Specimen (specimen processing area) The kit does not contain extraction. It is recommended to use RNA extration kits produced by QIAGEN and Roche or Hunan Runmei Gene Technology Co., Ltd. to extract viral RNA. The specific operation is in accordance with its kit instructions. 

3.Loading Add 5μl each of the processed specimen nucleic acid, COVID-19 Negative Control, and COVID-19 Positive Control to the PCR reaction tubes/plates to which the reaction mix has been added. The final volume is 25ul. Cap the tube tightly or seal the membrane, and then immediately amplify by low- speed centrifugation and a fluorescence PCR detector. 

4. PCR Amplification (nucleic acid amplification area) (1) Take the PCR reaction tube to be detected, place it in the corresponding position of the real-time fluorescent quantitative PCR instrument sample tank, and record the placing order. (2) Set the relevant parameters of nucleic acid amplification in the following table for PCR amplification Note: 1) the fluorescence collection is set at "step 3: 55 ℃, 30s". The detection channels were fam, Rox and Cy

5. Fam channel was orf1ab gene, and Rox channel was N gene. Cy5 channel was an internal reference gene, and the reaction system was set at 25 μ L. 2) ABI Series Fluorescent PCR instruments do not choose Rox calibration, but choose none as quench group.

POSITIVE JUDGEMENT VALLUE OR REFERENCE INTERVAL 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 threshold line. 

The Validity of The kit 

·The COVID-19 Positive Control: FAM and ROX channels have typical S-type amplification curves and values ≤32. The Internal Control channel (Cy5) have a typical S-type amplification curves. 

·The COVID-19 Negative Control: FAM, ROX and Cy5 channels have no Ct value or Ct value>38. Note: The above conditions must be met at the same time, otherwise this experiment is invalid and need to be repeated. 

·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. 

THE SPECIMEN RESULT JUDGMENT 

1. If the test specimen detects a typical S-type amplification curve in the FAM and ROX channels and the Ct value is ≤38, and there is a typical S-type amplification curve in the Internal Control channel (Cy5), the specimen can be judged to be COVID-19 positive.

2. If the test specimen has no amplification curve or Ct value> 38 in the FAM and ROX channels, and there is a typical S-type amplification curve in the Internal Control channel(Cy5), the specimen can be judged to be COVID-19 negative. 

3.If the test specimen only has a Ct value ≤38 in a single channel of the FAM or ROX channel, and there is a typical S-type amplification curve in the Step Temperature Time cycles 1 50℃ 10min 1 2 95℃ 3min 1 3 95℃ 10s 40 55℃ 30sInternal Control channel (Cy5), the results need to retest. If the results repeated are consistent, the specimen 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(Cy5), 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, ROX, and cy5 channels of the test specimen, it means that there is a problem with the quality of the specimen or a problem with the operation. If the result is invalid, you should find and eliminate the cause, collect the specimen again, and repeat the test (if the test result is still invalid, please contact the company). 

LIMITATIONS OF INSPECTION METHODS

The test results of this kit are for clinical reference only. The clinical diagnosis and treatment of patients should be considered in conjunction with their symptoms, signs, medical history and other related conditions. False negative results may occur when the concentration of the detected nucleic acid in the specimen is below the minimum detection limit of this kit. Improper handling of the tested specimen during collection, transportation, storage, and processing can easily result in RNA degradation and false negative results. When specimens are cross-contaminated during collection, transportation, storage, and processing it is ealy to get false positive results. 

PRODUCT PERFORMANCE INDEX

LOD: The limit of detection is 500 copies/mL Precision: Coefficient of variation (CV%) of precision Ct value within batch ≤3%. Specificity: No cross-contamination between this kit and positive specimens such as human coronavirus HCoV-NL63, Human coronavirus HCoV-0C43, SARS Coronavirus, MERS coronavirus, Influenza A virus, Influenza B virus Yamagata type, Victoria, H1N1 influenza virus, H3N2 influenza virus, H5N1 influenzavirus, H7N9 influenza virus, Respiratory syncytial virus A, Adenovirus(type 2), Adenovirus(type 2), Mycoplasma pneumoniae, Chlamydia pneumoniae, Pertussis, Streptococcus pneumoniae, Rhinovirus(type A)and so on.

PRECAUTIONS 

1. The entire detection process should be performed strictly in accordance with the requirements of this manual in the reagent preparation area, specimen processing area, and PCR amplification area, and the experimental clothes, instruments, and consumables in each area should be used independently and cannot be mixed The experimental tips use filter tips, the specimen processing area shouid be equipped with a biological safety cabinet the specimen processing should be performed in the biological safety cabinet, and three areas should be equipped with ultraviolet sterilization devices. 

2. To avoid RNA degradation, the specimen processing process should be operated at 0-4℃, and the test should be performed immediately after the experiment is completed. Utensil consumables used in specimen 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 specimen 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 instruction, and different batches of reagents cannot be mixed. 

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

【Instruction Approval and Revision Date】 

Approval Date:15 May,2020 Revision Date: NA Date of Issue:15 May,2020

【Index of Symbols】 Wenzhou Astock Biotechnology Co., Ltd. 

Address:(Room 1208, Building 1,Incubator, National University Science Park) No. 38,Dongfang Road,Wenzhou,Zhejiang,China.