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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, and instantaneously centrifuged. Remove the kit from the refrigerator when it is used again, shake briefly, and centrifuge instantly.

1. Sample handling (Sample handling area)

Add 10μL of internal control into 200μL of sample, positive control and negative control, respectively. Use nucleic acid extraction reagent (Beijing Changping Medical Device Filing NO. 20200008) of Beijing Applied Biological Technologies Co., Ltd. For nucleic acid extraction.

2. Reagents preparation (reagent preparation area):

1) Reaction system preparation: Take out the reagent and thaw the reagent completely. Invert the mixture 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, as follows. 

 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.

3. Loading (Sample handling area)

5μL of the extracted sample nucleic acid, positive control and negative control are added to the A and 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.

4. PCR amplification assay (Application assay area)

For the detection channel of tube A, the target channel of FAM and VIC is corresponding to ORF1ab gene and N gene respectively, and the internal channel is CY5. For the detection channel of tube B, the target channel of FAM is corresponding to E gene, and the internal channel is CY5.

1) ABI 7500: "Quencher Dye" and "Passive Reference" are set to “None” for the ABI 7500 instrument. Set the Positive Control, Negative Control, and Sample (Unknown) in order in which the samples correspond, and set the sample name in the

“Sample Name” column.

2) Roche Light Cycler 480Ⅱ , Roche Cobas z 480: Select "New Experiment" and select the target channel FAM, VIC, CY5 for sample detection in the "Detection format" drop-down menu in the "setup" panel. Set "Reaction Volume" to 20. The instrument parameter settings are as follows (the parameters not listed, select the instrument default value).

5. Threshold setting

After PCR reaction, manually or automatically adjust the Baseline and Threshold Value according to the IFU and fluorescence curve. If it is a manual adjustment, the Baseline is recommended to be set to be 3 and 15, while the threshold value can be determined according to fluorescence values of different instruments, take the point just above the highest fluorescence signal of normal negative control as the threshold value setting methods, or adjust according to the noise of the instrument. Click Analysis to automatically get the sample Ct value.

6. Experimental validity judgment

Each control of the kit must meet the following requirements, otherwise the experiment is invalid.

Note: the above two items should be met at the same time in an experiment, and have a typical s-shaped curve; Otherwise, this experiment is invalid.

[Cut-off Value]

According to the results of 100 oropharyngeal swab samples and 100 sputum samples, and with the ROC curve method, the Cut-off value of the OFR1ab, N and E gene of this kit are Ct = 38.

[Interpretation of Test Results]

1.The amplification curve of internal control (CY5) show a typical S curve and Ct≤38, interpretation of the results of target genes is subjected to the following conditions

Interpretation of results:According to the results of ORF1ab and N gene in tube A, and the result of E gene in tube B, interpretation as follows:

1) If any two genes of the detected genes are positive, the 2019-nCoV is positive;

2) If only one or none of the detected genes is positive, the 2019-nCoV is negative.

Note 1: If the target concentration is too high, it is normal that the internal channel may not be amplified with S shaped amplification curve in the corresponding tube with a typical S amplification curve for FAM or VIC in tube A, or FAM in tube B.

Note 2: If any two of target genes get Ct≤ 38, the 2019-nCoV is positive. If any two of target genes get Ct≥ 40, the 2019-nCoV is negative. If the other one 38<Ct<40, a retest is not necessary.

Note 3: If Ct ≥ 40, or show no value, interpretation the results of target gene is negative.

2. If all of the Ct values of FAM, VIC and CY5 channels are more than 38 or there is no obvious typical S amplification curve:

1) There is/are substance(s) in the sample that inhibit the PCR reaction. It is recommended to dilute the sample for being re-tested.

2) The process of nucleic acid extraction is abnormal, so it is suggested to re-extract nucleic acid for re-test.

3) This sample was NOT a qualified sample at the time of sampling, or degraded during transportation and storage.