Product Detail
This kit shall only be used for IVD emergency capacity for 2019-nCoV and auxiliary diagnosis of related cases with Novel Coronavirus infections, which can’t be used as a routine IVD in clinic.
Features
● 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.
● Powerful: Diagnosis of related cases with Novel Coronavirus infections
Technical Parameter
Name |
Multiple Real-Time PCR kit for Detection of 2019-nCov |
Packing Specification |
24 tests/kit, 48 tests/kit |
Target Region |
ORF1ab, N, E |
Applicable Sample |
Sputum, Oropharyngeal Swab |
Limit of Detection |
200 copies/ml |
Total Coincidence Rate |
99.55% |
Ct value (CV,%) |
≤5.0% |
Positive Coincidence Rate |
99.12% |
Negative Coincidence Rate |
100% |
Storage Conditions and Expiration Date |
Stored at -20±5℃, and is valid provisionally for 12 months. |
Internal Control |
Yes |
Catalogue Number |
CT8223-24T, CT8223-48T |
Certification |
CE, also in WHO list |
Specimens |
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

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.
Components |
Reaction system A |
Reaction system B |
Nuclease-free water |
2 µL×N |
2 µL×N |
Nucleic acid amplification reaction solution |
10 µL×N |
10 µL×N |
20×Reverse transcriptase |
1 µL×N |
1 µL×N |
10×reaction solution (A/B) |
2 µL×N |
2 µL×N |
Total volume |
15 µL×N |
15 µL×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.
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.
Number of Cycles |
Temperature |
Reaction time |
|
1 |
45 ℃ |
10 min |
|
1 |
95 ℃ |
5 min |
|
45 |
95 ℃ |
15 sec |
|
|
60 ℃ |
45 sec |
Fluorescence Collection |
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).
Program |
Cycles |
Target (℃) |
Hold (hh:mm:ss) |
Acquisition Mode |
Analysis Mode |
1 |
1 |
45 ℃ |
10 min |
None |
None |
2 |
1 |
95 ℃ |
5 min |
None |
None |
3 |
45 |
95 ℃ |
15 sec |
None |
Quantification |
60 ℃ |
45 sec |
Single |
|
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.
|
|
Negative control |
Positive control |
Tube A |
FAM channel (ORF1ab |
No Ct |
Ct≤38 |
VIC channel (N gene) |
No Ct |
Ct≤38 |
|
CY5 channel (IC) |
Ct≤38 |
—— |
|
Tube B |
FAM channel (E gene) |
No Ct |
Ct≤38 |
CY5 channel (IC) |
Ct≤38 |
—— |
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
|
Detection channels |
Interpretation the results of target genes |
|
FAM |
VIC |
||
Tube A |
Ct≤38 |
Ct≤38 |
With a typical S amplification curve, ORF1ab gene (FAM) and/or N gene (VIC) is positive. |
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. |
|
Ct≥40 |
Ct≥40 |
The corresponding target gene is negative. |
|
Tube B |
Ct≤38 |
--- |
With a typical S amplification curve, E gene is positive. |
38<Ct<40 |
--- |
With a typical S amplification curve, re-test the E gene of the sample again. If the Ct value<40, the E gene is positive; if the Ct value≥40, the E gene is negative. |
|
Ct≥40 |
--- |
The E gene is negative |
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.
FAQ
In this kit, primers and probes are designed to the conserved and specific regions of the ORF1ab, N and E gene of 2019-nCoV, respectively. After the purification of sample RNAs, they are detected using fluorescence PCR method.
The kit uses real-time fluorescence quantitative PCR technique. 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 by the real-time fluorescence quantitative PCR instrument (the number of cycles experienced by the fluorescence signal in each reaction tube when it reaches the set threshold). FAM and VIC fluorophores are labeled to ORF1ab gene and N gene probes, respectively in reaction solution A, and FAM fluorophore is labeled to E gene probe in reaction solution B. By using one test with two 10× reaction solutions, qualitative detection of the above three genes of 2019-nCoV can be performed simultaneously.
The kit is provided with an internal control (IC), which is pseudo-virus containing unrelated genes fragments, and is labeled with a CY5. It is used to monitor the extraction process and possible inhibitory factors in the reaction system, indicating false negative 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
|
Detection channels |
Interpretation the results of target genes |
|
FAM |
VIC |
||
Tube A |
Ct≤38 |
Ct≤38 |
With a typical S amplification curve, ORF1ab gene (FAM) and/or N gene (VIC) is positive. |
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. |
|
Ct≥40 |
Ct≥40 |
The corresponding target gene is negative. |
|
Tube B |
Ct≤38 |
--- |
With a typical S amplification curve, E gene is positive. |
38<Ct<40 |
--- |
With a typical S amplification curve, re-test the E gene of the sample again. If the Ct value<40, the E gene is positive; if the Ct value≥40, the E gene is negative. |
|
Ct≥40 |
--- |
The E gene is negative |
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.
They are 2 ways we can detected this situations: NAAT and Antigen.
(Comes from CDC of Los Angeles)