Method for detecting novel coronavirus virus based on real-time fluorescence RT-RAA

Abstract

The invention discloses a method for detecting novel coronavirus virus based on real-time fluorescence RT-RAA. The method comprises the following steps: step 1, reserving a sample; 2, extracting nucleic acid; step 3, constructing standard plasmids; step 4, carrying out RT-RAA detection; step 5, testing sensitivity and specificity; step 6, carrying out a precision test, wherein in the step 1, a throat swab, a nasopharynx extract or a respiratory tract extract, a deep expectoration solution, a bronchial lavage solution, an alveolar lavage solution, a blood specimen, a serum specimen and an eye conjunctival swab are manually collected. According to the method for detecting the novel coronavirus virus based on the real-time fluorescence RT-RAA, a novel real-time fluorescence RT-RAA method fordetecting SARS-CoV-2 is constructed based on a recombinase mediated isothermal nucleic acid amplification technology, and compared with other molecular diagnosis methods, the method is high in detection sensitivity, easy, convenient and rapid to operate and suitable for large-scale industrial production. No special equipment is needed, the result is accurate, clinical sample detection can be met,and the method is particularly suitable for mobile emergency detection of resource-deficient areas or airports, schools and other units.

Description

technical field [0001] The invention relates to the technical field of novel coronavirus detection, in particular to a method for detecting novel coronavirus based on real-time fluorescence RT-RAA. Background technique [0002] Novel coronavirus-severe acute respiratory syndrome coronavirus 2 is a pathogen that has not been found in humans before and can spread rapidly between humans; It is spreading worldwide and constitutes a public health emergency of international concern. As of June 14, 2020, 213 countries around the world have reported cases of new coronary pneumonia, with a total of 7,859,593 confirmed cases of new coronary pneumonia and a cumulative death toll of 4,321.68 million, and Confirmed cases are still increasing; there is no effective treatment for COVID-19, and the current methods are mainly isolation treatment and symptomatic support; therefore, strengthening the rapid detection of pathogens in COVID-19 laboratories is the key to effective monitoring and d...

AI Technical Summary

Problems solved by technology

[0003] Nucleic acid detection technology is the most direct and essential etiological evidence, and is the gold standard for the diagnosis of SARS-CoV-2 infection, which is superior to other clinical manifestations, clinical signs, and empirical interpretation of CT detection; it is currently used to detect SARS-CoV-2 The conventional method is to use real-time fluorescent RT-PCR, which can detect SARS-CoV-2 from nasal or throat swabs, sputum, or bronchoalveolar lavage fluid; however, real-time fluorescent RT-qPCR is costly and expensive, Requires a large amount of laboratory infrastructure and training, which hinders their use in areas with limited resources, such as rapid detection in areas with poor medical facilities or densely populated frontlines of prevention and control such as ports, airports or stations. In addition, long-term The process increases the risk of further spread of SARS-CoV-2 and prevents widespread testing of all potential contacts; The entire gene sequence of the virus is completely sequenced and compared with the known virus sequence to diagnose the existence of SARS-CoV-2. Although NGS has many advantages, including the ability to analyze the source of virus evolution, pathogenic pathological mechanism and virus mutation However, due to the limitation of NGS technology, it takes longer time, requires expensive next-generation sequencing equipment and strict operating environment, it is doomed that high-throughput sequencing technology can only be used as a supplement to real-time fluorescent RT-PCR. It is even less suitable as an emergency rapid screening method. Therefore, neither the above-mentioned methods are suitable for immediate testing in areas or units with relatively poor conditions;

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