2019-nCoV Multiplex RT-qPCR Detection Kit
Catalog No.: V2001 Size: 48 tests / kit
This kit uses real-time fluorescence quantitative PCR technology to qualitatively detect the 2019 Novel Coronavirus (2019-nCoV) in human nasopharyngeal swab samples.
In this kit, specific primers and probes were designed with conserved sequences of ORF1ab gene and N gene of the 2019-nCoV as the target regions, and One-step RT-PCR (RT-qPCR, reverse transcription and real-time fluorescence quantitative PCR) was used to detect the 2019-nCoV RNA by fluorescence signal changes.
The detection results of this kit are for scientific research only and shall not be used as the standard of clinical diagnosis.
The kit consists of the following components:
The name of the reagent
2019-nCoV PCR Buffer
Primers, Probes, Buffer, dNTPs
2019-nCoV Enzyme Mix
Reverse transcriptase (M-MLV), Hotstart Taq DNA polymerase
2019-nCoV Positive Control
Recombinant plasmid containing target fragment
2019-nCoV Negative Control
1) The above components with different batches shall not be used interchangeably.
2) Required reagent: nucleic acid extraction reagents.
Storage conditions & expiry date
1. The kit should be stored at -20±5°C away from light, valid for 6 months.
2. Store at 2-8°C after opening and use within 7 days; Avoid repeated freezing-thawing, repeated freezing-thawing should not exceed 5 times.
3. Dry ice shall be used for transportation to keep low temperature, and the transportation time shall not exceed 5 days.
ABI7500, ABI StepOne.
Requirements of sample
1. Applicable sample type: nasopharyngeal swabs.
2. Sample collection: please refer to the protocol in the "Microbiology Specimen Collection Manual".
3. Sample preservation and transportation: samples collected shall be submitted for inspection in time, and those detected within 24 hours shall be stored at 4°C. The samples shall be transported by foam box sealed with ice.
1. Reagent Preparation (PCR Reagent Preparation Area)
1.1 Thaw all components in the kit at room temperature until the temperature is balanced to room temperature;
1.2 Prepare the mixture according to the number of samples to be tested, negative control and positive control in a RNase-free centrifuge tube. The dosage for one test is as follows:
Volume (μl Per Reaction)
2019-nCoV PCR Buffer
2019-nCoV Enzyme Mix
Mix the above mixture thoroughly, and centrifuge briefly. Transfer mixture of 16.5 μl into different PCR reaction tubes. Then, move to the Sample Preparation Area.
2. Sample Preparation (Sample Preparation Area)
2.1 The samples should be extracted according to the corresponding requirements
and procedures of viral RNA extraction kits. The 2019-nCoV Negative Control in the kit should be processed synchronously with the samples for monitoring the extraction process and environment.
2.2 Add 13.5 μl of sample RNA, extracted Negative Control products and 2019-nCoV Positive Control into the PCR mixtures of corresponding test wells. The total volume is 30 μl. Cap the reaction tubes tightly. Then, move to the Detection Area.
Chart 1 Layout diagram of PCR (96 wells)
2019-nCoV PCR Mixture
3. RT-qPCR Amplification (Detection Area)
3.1 Put the reaction tubes on a PCR instrument after a short centrifugation. Set the positive control, negative control and test samples in the corresponding order, and set the sample name.
3.2 Settings of detection fluorescence: 2019-nCoV-ORF select FAM channel, 2019-nCoV-N select HEX channel.
3.3 Setting cycling parameters
Chart 2 Reaction Procedure
4. Data Analysis：
4.1 Setting analysis conditions:
Adjust the start Value, stop Value and Threshold Value of the Baseline according to the analyzed image (Start value: 3-15, End value: 5-20, the amplification curve of the negative control should be straight or below the threshold line). Click ”Analysis” to obtain the analysis result automatically, and read the
detection result in the "Report" window.
4.2 Quality standard：
negative control：FAM and HEX channel No Ct or Ct > 37.
positive control：FAM and HEX channel Ct ≤ 33.
4.3 Analyzing the data of the samples：
4.3.1 FAM or HEX channel Ct ≤ 37, it is reported as Positive for the corresponding gene.
4.3.2 FAM or HEX channel Ct > 40, it is reported as Negative for the corresponding gene.
4.3.3 FAM or HEX channel 37 < Ct ≤ 40, it is reported as Suspected Positive. It is suggested to re-collect sample and test again. If the Ct value is still ≤ 40 and the amplification curve has obvious peaks, the sample is judged to be positive for the corresponding gene; otherwise, the corresponding gene is negative.
Criteria for the 2019 Novel Coronavirus (2019-nCoV) (Refer to relevant information released by CDC)
If both 2019-ncov-orf and 2019-ncov-n genes are tested positive, it could be judged that the sample is positive for novel coronavirus. If only one gene tests positive, the new coronavirus is suspected. Negative results do not preclude novel coronavirus infection. Factors that may produce false negatives need to be excluded, including poor sample quality, early or late sample collection, low virus content, incorrect storage, transportation and handling of sample, and virus mutation, etc.