Multiple qPCR kit for detection of 2019-nCoV (1 tube)

The kit is a TaqMan probe real-time fluorescent PCR to qualitatively detect ORF1ab, N and E gene nucleic acid of 2019-nCoV (SARS-CoV-2) in nasopharyngeal swab, oropharyngeal swab, alveolar lavage fluid, saliva and sputum in patients with respiratory infection symptoms and close contacts.

Product Detail

The World Health Organization (WHO) guides that antigen-detecting rapid diagnostic tests (Ag-RDRs) can offer a faster and less expensive way to diagnose active SARS-CoV-2 infection than nucleic acid amplification tests (NAATs), and WHO also recommends that Ag-RDTs meeting minimum performance requirements can be used for primary case detection, contact tracing, during outbreak investigations and to monitor trends of disease incidence in communities.

Multiple qPCR kit for detection of 2019-nCoV (1 tube) (3)


● Comprehensive: Three target gene detects in one test

● compatible: Adaptive to common equipment with CY5, FAM, VIC/HEX channels.

● Excellent performance: High sensitivity and specificity, LOD = 200 copies/ml.

Technical Parameter

Packing Specification

50 tests/kit, 100 tests/kit

Target Region

ORF1ab, N, E

Applicable Sample

Sputum, Oropharyngeal Swab

Limit of Detection

200 copies/ml

Total Coincidence Rate


Ct value (CV,%)


Positive Coincidence Rate


Negative Coincidence Rate


Storage Conditions and Expiration Date

Stored at -20±5℃, and is valid provisionally for 12 months.

Internal Control


Catalogue Number

A7793YF-50T, A7793YF-100T




Nasopharyngeal swab, Oropharyngeal swab, Alveolar lavage fluid, Saliva and sputum

Applicable Instrument

ABI 7500, Roche Light Cycler 480Ⅱ , Roche Cobas z 480, SLAN-96P Real-Time PCR System

Test Procedure

Multiple qPCR kit for detection of 2019-nCoV (2 tube) (1)

1. Nucleic Acid Extraction

Operation should be carried out according to the manual of extraction kit.

2. System Preparation:

1) 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 + positive control + negative control + 1) are prepared for reaction systems, respectively, as follows.


Volume for 1 reaction system

Volume for N reaction system

Nucleic acid amplification reaction solution Mix (A7793YF)

18 µL

18 µL * N

Enzyme mixture

2 µL

2 µL * N

Total volume

20 µL

20 µL * N

2) Reaction distribution: The reaction solution was mixed and centrifuged, and each tube was dispensed in an amount of 20μL in a PCR tube suitable for a fluorescence PCR apparatus.

3. Loading

5μL of the extracted sample nucleic acid, positive control nucleic acid and negative control nucleic acid are added to the reaction systems, and the total reaction volume is 25μL. Fasten the tube cover and move it to the amplification test area after a few seconds of centrifugation.

4. PCR Amplification Assay

1) Put the PCR reaction tube into the fluorescent PCR amplification instrument for amplification detection.

2) Cycle parameter setting:


Number of cycles


Reaction time




10 min




30 sec




5 sec


30 sec

Fluorescence Collection

3) Detection settings:

The detection channels are set to FAM, VIC ,ROX and CY5, corresponding to ORF1ab, N gene, and E Gene, RNase P internal control, respectively. "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.

For X-POCH16, the operation and program are as follows:

1) After the self-test is complete, open the lid and put the PCR reaction tubes into the designated positions in the instrument.

2) Start by selecting the "Exper." option. Select the "All" option or manually select the reaction area on the left side of the screen.

3) Select the "LOAD" option; select the test program; click “DONE” and the “RUN”. The program takes 30min42s to complete.

The detection channels of the Default program are set to FAM, VIC, ROX and CY5, corresponding to ORF1ab, N gene, and E Gene, RNase P internal control, respectively.

The cycle parameter of the Default program are as follows:


Number of


Reaction time
















5. Threshold Setting

According to the analyzed image, adjust the Start value, End value of Baseline and Threshold value (Start value and end value is recommended to be set to be 3 and 15 respectively, and the amplification curve of the negative control is adjusted to be flat or lower than the threshold Line), click Analysis to automatically get the analysis the sample Ct value. View the results in the Report interface.

6. Quality Control Standard

Each control of the kit must meet the following requirements with ‘S’ curve, otherwise the experiment is invalid.

Detection channels

Negative control

Positive control


No Ct



No Ct



No Ct



No Ct


【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 genes E Gene of this kit are Ct = 38


How does this kit work?

In this kit, primers and probes of real-time fluorescent PCR technology are designed to the conserved and specific regions of the ORF1ab, N and E gene of 2019-nCoV respectively. During PCR amplification, the probe binds to the template, and the 5'-end reporter group of the probe is cleaved by the Taq enzyme (5'→3' exonuclease activity), thereby moving away from the quenching group to generate a fluorescent signal. The real-time amplification curve is automatically plotted based on the detected fluorescence signal, and the sample Ct value is calculated. FAM , VIC and ROX fluorophores are labeled to ORF1ab gene ,N gene and E gene probes. By using one test, qualitative detection of the above three genes of 2019-nCoV can be performed simultaneously.

The kit is provided with an internal control targeting the RNase P gene to monitor clinical samples collection, handling, extraction and RT-PCR process to avoid false-negative results. The internal control is labeled with a CY5 fluorescent group.

How to interpret test results?

1. Analyze and interpret test results when the instrument is normal, and the positive control, the negative control and the internal control test result meet the quality control standard.

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

Detection channels

Interpretation the results of target genes



(N gene)


(E gene)




With a typical S amplification curve, the Ct value is≤38, the corresponding target gene is positive.

38 < Ct < 40

38 < Ct < 40

38 < Ct < 40

With a typical S amplification curve, re-test the corresponding target gene of the sample again.

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




The corresponding target gene is negative

Interpretation of results for 2019-nCoV:

According to the results of ORF1ab, N gene and E gene, interpretation as follows:

1) If TWO or THREE 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: The amplification curve of the positive sample should be with a typical S curve. However, if the target concentration is too high, the internal standard control may not be amplified and the sample can be directly judged as positive. 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 Ct≥40, or show no value, interpretation the results of target gene is negative.

3. If all of the Ct values of FAM, VIC, ROX 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.

How many ways can we detect whether we had infected with the COVID-19/ SARS-CoV-2

They are 2 ways we can detected this situations: NAAT and Antigen.

Neutralization Antibody Rapid Test Cassette (5)

 (Comes from CDC of Los Angeles)

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