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A multiplex fluorescent rt-pcr detection reagent for detection of 2019 novel coronavirus imported at ports

A RT-PCR, coronavirus technology, applied in the field of inspection and quarantine, can solve the problems of short time and false negatives of nucleic acid detection methods

Active Publication Date: 2021-05-18
BEIJING CUSTOMS DISTRICT P R CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the sudden outbreak of novel coronavirus pneumonia, nucleic acid detection methods have been used in clinical practice for a short period of time, and sufficient performance verification and clinical application evaluation have not yet been carried out. In the sample detection, there have been false negatives in which the nucleic acid detection results are inconsistent with the clinical diagnosis.

Method used

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  • A multiplex fluorescent rt-pcr detection reagent for detection of 2019 novel coronavirus imported at ports
  • A multiplex fluorescent rt-pcr detection reagent for detection of 2019 novel coronavirus imported at ports
  • A multiplex fluorescent rt-pcr detection reagent for detection of 2019 novel coronavirus imported at ports

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 detects the design of special primers and probes for 2019 novel coronavirus

[0051] All SARS-Cov-2 genome sequences from all over the world were obtained from GenBank and GISAID. The database has a total of 3356 entries marked as genome, and each motif is compared to 3148 genome sequences respectively. The parameter value=0.01 (99.99% credible) Degree), the sequence identity is more than 90%, except for the whole gene fragment less than 29000bp and the same whole genome sequence, the sequence comes from 40 countries and regions, MEGA X software evolution analysis, a total of 413 genomes. Obtain 413 full lengths of the RdRP gene, N gene, and S gene of the 2019 novel coronavirus genome, respectively, and obtain the conserved gene sequences of the SARS-Cov-2 RdRP gene, N gene, and S gene, according to the general principles of primer and probe design , use the software PrimerExpress 3.0 to design primers and TaqMan probes, then compare the designed primers an...

Embodiment 2

[0052] Embodiment 2 detects the composition of the multiplex RT-qPCR detection kit of 2019 novel coronavirus

[0053] (1) qPCR reaction solution, which includes the sense primer shown in the sequence listing SEQ ID No.1 / 3 / 5 / 7 / 9, the antisense primer shown in the sequence listing SEQ ID No.2 / 4 / 6 / 8 / 10 , the fluorescent probe shown in the sequence table SEQ ID No.11-15, the 5' end of the probe is labeled with a reporter fluorescent group containing FAM, CY5, HEX, VIC, TEXAS RED reporter fluorescent dyes, and the 3' end is labeled with quenching fluorescence Group BHQ1;

[0054] (2) Negative control: Negative control: Use a flocked sterile long cotton swab to quickly wipe the palatal arches, pharynx, and tonsil secretions on both sides of the 2019-nCoV-infected person, insert the cotton swab into the culture tube, shake the culture tube for 1 minute, and take 200 μL throat swab culture fluid, extract RNA, dissolve in ddH 2 O;

[0055] Further, the above qPCR reaction solution a...

Embodiment 3

[0056] Example 3 Multiplex RT-qPCR specificity test for detection of 2019 novel coronavirus

[0057] The specific process includes the following steps:

[0058] 1. Sample source

[0059] 2019 novel coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-Cov-2), acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus, SARSr-CoV) and Middle East respiratory syndrome coronavirus (Middle East respiratory syndrome coronavirus, MERSr- CoV) was kept by the Beijing Customs Laboratory.

[0060] 2. Extraction of sample RNA

[0061] Extract the total plant DNA from the sample in step 1 with a DNA extraction kit (purchased from Qiagen), see the kit instructions for specific operations; (1) extract sample RNA;

[0062] Get the sample carrying 2019 novel coronavirus, extract RNA through RNA extraction kit (QIAamp Viral RNA MiniKit, purchased from Qiagen company), also can use the RNA method known in the prior art to extract;

[0063] 3. Specific ...

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Abstract

The invention discloses a multiplex fluorescent PCR primer, probe and kit for identifying 2019 novel coronavirus, the primers are shown in the sequence SEQ ID No.1 to SEQ ID No.10, and the probe is shown in the sequence SEQ ID Shown in No.11 to SEQ ID No.15, the present invention also provides a detection kit containing the above-mentioned primers and probes and a detection method thereof, which can achieve the purpose of accurately detecting SARS-Cov-2 in the sample to be tested. The present invention The kit is sensitive, fast and accurate, and it can detect the 2019 novel coronavirus. This technology has great application prospects for the detection and monitoring of overseas 2019 novel coronavirus at various ports.

Description

technical field [0001] The invention relates to multiple fluorescent RT-PCR detection kits and oligonucleotides for detecting 2019 novel coronavirus imported at ports, and belongs to the field of inspection and quarantine. Background technique [0002] The 2019 novel coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-Cov-2) is a single-stranded positive-strand RNA virus with a diameter of 60-140nm in the genus of coronavirus, and it is a newly discovered member of the genus of β-coronavirus. It has been reported in 200 countries and regions have reported confirmed cases. Common signs of people infected with 2019-nCoV include respiratory symptoms, fever, cough, shortness of breath and dyspnea. In more severe cases, the infection can lead to clinical manifestations of pneumonia, severe acute respiratory syndrome, renal failure, and even death. [0003] The prevention and control of SARS-Cov-2 is at a critical stage. Nucleic acid detection, as the main means o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 张锡全林劲松刘兴亮张利峰邓丛良赵靖敏
Owner BEIJING CUSTOMS DISTRICT P R CHINA