Novel coronavirus nucleic acid quantitative detection kit based on micro-droplet digital analysis

Abstract

The invention discloses a novel coronavirus nucleic acid quantitative detection kit based on micro-droplet digital analysis. The novel coronavirus nucleic acid quantitative detection kit is characterized by comprising O-crRNA targeting the ORF1ab gene 6513-6537nt of targeted SARS-CoV-2 RNA, N-crRNA targeting the N gene 29214-29238nt of targeted SARS-CoV-2 RNA, Cas13a protein, a single-chain RNA report probe and a reaction buffer solution. The detection method does not need the steps of reverse transcription or nucleic acid sequence amplification, so that the pollution risk possibly caused by sample loss, amplification deviation and nucleic acid replication products can be avoided; the operation steps are simple, expensive thermal cyclers are not needed, and the detection time is not more than 1 hour; SARS-CoV-2 RNA single copy level detection and absolute quantification can be realized, and internal reference correction and a standard curve establishment are not needed.

Description

technical field [0001] The invention relates to the field of viral nucleic acid detection, in particular to a novel coronavirus nucleic acid quantitative detection kit based on micro-droplet digital analysis. Background technique [0002] The novel coronavirus SARS-CoV-2 is the causative agent of the global outbreak of novel coronavirus pneumonia (COVID-19). The methods currently used to detect SARS-CoV-2 mainly include: virus isolation and culture and electron microscope observation, immunological detection and molecular biological detection (including nucleic acid detection and sequencing). Among them, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) detection in molecular biology detection methods is currently one of the gold standards for the diagnosis of new coronary pneumonia. However, there are also some problems in the clinical use process: when the sample virus nucleic acid content is low, there is interference of PCR inhibitory comp...

AI Technical Summary

Problems solved by technology

However, there are also some problems in the clinical use process: when the sample virus nucleic acid content is low, there is interference of PCR inhibitory components, virus mutation and primers, probe binding ability decline and other factors exist, the positive rate of qRT-PCR detection is low and needs to be tested. Repeated testing or false negatives resulting in missed testing

These steps will also bring a series of problems, including sample loss caused by incomplete reverse transcription, complexity of primer and detection probe design, amplification bias caused by sequence duplication process errors, and false positive risk caused by amplification product contamination

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