Digital PCR-based novel coronavirus nucleic acid quantitative detection kit and application

A detection kit, a technology for coronavirus, applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems of false negatives, variations, and uninvolved diagnostic methods.

Active Publication Date: 2020-07-10
南京实践医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Multi-day clinical test results show that this diagnostic method has a high rate of false negatives, and often requires multiple nucleic acid tests before it can be finally verified
At the same time, there may be mutations in this new type of coronavi

Method used

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  • Digital PCR-based novel coronavirus nucleic acid quantitative detection kit and application
  • Digital PCR-based novel coronavirus nucleic acid quantitative detection kit and application
  • Digital PCR-based novel coronavirus nucleic acid quantitative detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0087] Implementation case 1: Using the positive control of pseudovirus to detect the sensitivity of the droplet digital PCR system

[0088] In this implementation case, the positive control (pseudovirus) of 2019-nCoV was used for a series of gradient dilutions, and the corresponding viral RNA was extracted from the diluted series of pseudovirus solutions, and then the corresponding droplet digital PCR detection was performed. In the case of satisfying a minimum of 3 microdroplets in digital PCR, the minimum detectable sensitivity of the ORF1ab gene of the 2019-nCoV pseudovirus is 5copies / reaction, and the minimum detectable sensitivity of the N gene is 3copies / reaction, so the present invention will detect the sensitivity It is defined as 5copies / reaction, which is much higher than the fluorescent PCR product approved by the State Food and Drug Administration (the lowest sensitivity is 100copies / reaction).

Embodiment example 2

[0089] Implementation Case 2: Clinical Sample Testing

[0090] Due to the particularity of the novel coronavirus pneumonia, the kit described in the present invention only collects throat swabs from 10 employees after returning to work for fluorescent RT-PCR detection and droplet digital PCR detection of 2019-nCoV. RT-PCR detection uses products that have been approved by the State Food and Drug Administration, and 2019-nCoV droplet digital PCR detection uses the method described in the present invention.

[0091] According to the digital PCR detection process of the present invention and the commercial RT-PCR kit detection method process, the detection results of throat swabs of 10 employees are shown in Table 3.

[0092] Table 3 Summary of 2019-nCoV digital PCR and fluorescent RT-PCR detection results

[0093] sample number Droplet digital PCR test results Fluorescent RT-PCR detection results A01 Negative Negative A02 Negative Negative A03 ...

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Abstract

The invention discloses a digital PCR-based novel coronavirus nucleic acid quantitative detection kit and an application. The reaction total system of the digital PCR-based novel coronavirus nucleic acid quantitative detection kit has 20 ul, comprising 10ul of 2x One-Step RT-ddPCR Supermix, 0.8ul of 25mM manganese acetate solution, 5ul of to-be-detected sample RNA, 1ul of ORFlab gene primer probeworking solution, 1ul of N gene primer probe working solution, 1ul of RPP30 gene primer probe working solution and 1.2ul of Nuclease-Free Water, wherein 5ul of negative control RNA extraction solutionand 5ul of positive control RNA extraction solution are adopted to replace the to-be-detected sample RNA in a negative control reaction system and a positive control reaction system respectively. Based on the innovative RNA one-step reverse transcription microdroplet type digital PCR technology, nucleic acid absolute quantification is carried out specific to highly conservative ORFlab gene and Ngene in the novel coronavirus (2019-nCoV) genome, so that the detection accuracy is improved, and the kit can be used for clinical assisted diagnosis and viral load analysis of novel coronavirus (2019-nCoV) infection, and has a wide clinical application value.

Description

technical field [0001] The invention relates to the technical field of novel coronavirus detection kits, in particular to a digital PCR-based nucleic acid quantitative detection kit for novel coronavirus and its application. Background technique [0002] The new coronavirus was named 2019-nCoV by the WHO, and was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses on February 12, 2020. The virus can cause severe acute respiratory infection (Severe acute respiratory infection, SARI), and its early symptoms are similar to common viral colds. As the disease progresses, patients will experience dyspnea, chest tightness, and even respiratory distress symptoms. Medical imaging examinations show multiple ground-glass opacities in the lungs, and lung consolidation may occur in severe cases. In view of the characteristics of highly contagious and clustered onset of the new coronavirus, timely screening of people in...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/701C12Q1/6851C12Q2563/159C12Q2521/107C12Q2545/101C12Q2537/16Y02A50/30
Inventor 张鹏唐春花谢珍邢宽倪海钰夏统前
Owner 南京实践医学检验有限公司
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