Novel coronavirus 2019-nCoV nucleic acid kit and virus nucleic acid acquisition method

Abstract

The invention discloses a novel coronavirus 2019-nCoV nucleic acid kit and a virus nucleic acid acquisition method. The method comprises the following steps: according to novel coronavirus 2019-nCoV ORF1ab and an N gene as amplification target areas, designing specific primers and fluorescent probes for detecting novel coronavirus 2019-nCoV nucleic acid in samples; and performing Digital PCR (polymerase chain reaction) absolute quantification on 2019-nCoV nucleic acid RNA (ribonucleic acid) by using a one-step method by using a prepared Super premix reaction liquid A together with Super premixreaction liquids B and C, and performing Digital PCR detection on RNA of a diluted positive virus sample. The kit and the method have the beneficial effects that by using the novel virus nucleic acidacquisition method, and together with a Digital PCR probe method, the nucleic acid positive detection rate of infection of the novel coronavirus 2019-nCoV can be increased, the process is ensured tobe specified and standardized, the influence of artificial non-standard operation upon detection results can be reduced, result reliability can be ensured, and meanwhile, false negative results causedby detection of sputum and throat swab sampling together with real-time fluorescent PCR methods can be avoided.

Description

technical field [0001] The invention relates to the field of detection kits, mainly a novel coronavirus 2019-nCoV nucleic acid kit and a method for collecting viral nucleic acids. Background technique [0002] The 2019 novel coronavirus (2019 novel coronavirus, 2019-nCoV) was named by the World Health Organization (WHO) on January 12, 2020. Reliable laboratory diagnosis is a top priority in promoting public health interventions. In acute respiratory infections, real-time PCR, or RT-PCR, is commonly used to detect pathogenic viruses in respiratory secretions. [0003] At present, the real-time fluorescent PCR method is mainly used to detect 2019-nCoV nucleic acid (development reading frame 1ab and nucleocapsid protein) in clinical practice for early diagnosis of cases, and the diagnosis can only be confirmed if it is positive. Sourced samples primarily consisted of sputum and nasal and throat swabs with or without viral transport media. For some samples with low viral load...

AI Technical Summary

Problems solved by technology

For some samples with low viral load abundance, the fluorescent PCR method often produces many false negative results due to the sensitivity. Patients who were positive for six throat swab nucleic acid tests not only increased the workload of medical staff, but also could easily become a source of infection again if these false-negative patients were not handled properly

The reason for the low detection rate is that in addition to the limitations of the fluorescent PCR method itself, more importantly, it is because of the characteristics of the new coronavirus itself that the virus level in the upper respiratory tract such as the pharynx is very low, so we need a more effective method. Virus sample collection method, combined with Digital PCR, can directly detect the absolute number of nucleic acid molecules, and its sensitivity can be as low as a single copy. It can accurately quantitatively detect low-abundance samples, which is expected to greatly improve the detection rate.

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