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Novel coronavirus SARS-CoV-2 detection and molecular typing method and kit

A molecular typing and sars-cov-2 technology, applied in the field of molecular biology detection, can solve the problem of not being able to provide SARS-CoV-2 subtype and mutation information

Active Publication Date: 2021-04-09
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these assays are faster, easier, less costly and less time consuming to confirm COVID-19 than NGS, they do not provide subtype and mutation information of SARS-CoV-2

Method used

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  • Novel coronavirus SARS-CoV-2 detection and molecular typing method and kit
  • Novel coronavirus SARS-CoV-2 detection and molecular typing method and kit
  • Novel coronavirus SARS-CoV-2 detection and molecular typing method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 is carried out based on the premise of the present invention, and a detailed implementation manner and specific operation process are given. The specific implementation and operation process described here are only used to explain the present invention, but the protection scope of the present invention is not limited to the following examples. The implementation and specific operation process of the embodiment of SARS-CoV-2 detection and molecular typing are described below with respect to the clinical use of the present invention in hospitals.

[0064] 1) Use a dedicated virus collection tube to collect patient respiratory specimens (nasopharyngeal swabs, throat swabs, saliva, sputum, and lower respiratory tract secretions).

[0065] 2) Extract the viral RNA in the sample according to the operating instructions of the QIAamp Viral RNA Mini kit, and add RNase inhibitors to the RNA preservation tube at the same time. (The above steps can be used to complete...

Embodiment 2

[0070] Embodiment 2, the performance measurement of kit

[0071] 1. Verification of accuracy

[0072] The RNA extracted from 5 isolated strains of SARS-CoV-2 after infection of cells and 6 clinical samples of positive patients were selected, and tested with reference to the method and steps of the above-mentioned Example 1. The results showed that Assay1 of these samples could accurately identify the three SARS-CoV-2 detection target genes, and these samples were determined to be positive for SARS-CoV-2; and according to the detection results of 9 marker mutation sites of Assay2-4, these samples were typed into 5 different GISAID types , including L, S, V, G and GR. At the same time, the PCR-sequencing method was used to sequence the nine marker mutation sites of these samples, and the results showed that the typing results of this method were consistent with those obtained by the PCR-sequencing method. In summary, it shows that the method has good detection and typing perfo...

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PUM

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Abstract

The invention provides a novel coronavirus SARS-CoV-2 detection and molecular typing method and a kit. The method utilizes the combination of one-step multiple RT-PCR and HRM analysis to simultaneously realize the detection and typing of SARS-CoV-2. The detection method comprises the following steps of: 1) extracting sample nucleic acid; 2) completing one-step multiple RT-PCR reaction in a special primer group and a reagent; and 3) analyzing and interpreting SARS-CoV-2 detection results and molecular typing results by HRM. The kit provided by the invention comprises multiple special primers for detecting SARS-CoV-2 and performing molecular typing on the SARS-CoV-2, and also comprises reaction components Master Mix (RNA reverse transcriptase, polymerase, amplification buffer, dNTP and EvaGreen fluorescent dye), positive control and negative control.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, relates to a pathogen detection method, in particular to a novel coronavirus SARS-CoV-2 detection and molecular typing method and a kit. Background technique [0002] Coronaviruses (Coronaviruses, CoVs) belong to the Coronaviridae family and are a large family of viruses that exist widely in nature and can infect humans and animals. The genome of coronavirus is relatively large, and its single-stranded positive-strand gene sequence is as long as 26-32kb, which is the RNA virus with the longest known sequence in nature. [0003] So far, Coronaviridae viruses only infect vertebrates, mostly causing respiratory and digestive tract symptoms in humans and animals, and other organ and nervous system symptoms have also been occasionally reported. Human coronavirus infection mainly includes upper respiratory tract, lower respiratory tract and gastrointestinal symptoms. Mild symptoms ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6858C12Q2600/156C12Q2600/16C12Q2521/107C12Q2531/113C12Q2527/107C12Q2563/107C12Q2537/143Y02A50/30
Inventor 彭俊平修乐山孙立莹
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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