Combination of primers and CRISPR sequence for detecting novel coronavirus gene, and application thereof

Abstract

The invention provides combination of primers and a CRISPR sequence for detecting novel coronavirus gene comprising two groups of primer pairs for RPA isothermal amplification for respectively detecting virus ORF1ab genes and N genes, and two crRNA sequences capable of respectively detecting RPA amplification products. The ORF1ab gene and the N gene can be detected at the same time through systemdetection, operation is easy and convenient, and each detection sample only needs to be detected through a single tube; the whole reaction process is carried out at the temperature of about 37 DEG C,a fine temperature control element and complex temperature change which are required by PCR amplification are not required, and the combination is suitable for a basic apartment to detect by using relatively cheap instant detection equipment; and the sensitivity reaches 10 copies / uL. Compared with a PCR technology, the advantages are that the speed is higher, an obvious detection signal can be obtained within 30 min, and whether the 2019-nCoV novel coronavirus nucleic acid result of a sample is positive or not can be rapidly judged.

Description

technical field [0001] The invention belongs to the technical field of genetic detection, and in particular relates to a detection system for detection of 2019-nCoV novel coronavirus ORFab1 gene and N gene and its application. Background technique [0002] Nucleic acid detection is the gold standard for non-invasive diagnosis of 2019-nCoV. Now 2019-nCoV nucleic acid detection generally adopts the real-time RT-PCR method. After the supply of detection kits increased, many cases found that the kits were negative. They have actually been infected with the new coronavirus, and the real-time PCR test takes a long time and is complicated to operate, making it difficult to meet the rapidly growing demand for investigation of suspected cases. Therefore, there is a demand for detection kits in terms of sensitivity, detection speed, price, and ease of operation. [0003] In recent years, a developed gene detection technology characterized by clustered regularly interspaced short pali...

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Problems solved by technology

[0004] On February 14, 2020, Professor Zhang Feng's team released the detailed operation process of using CRISPR / Cas13-based SHERLOCK technology to detect new coronaviruses. The essence is still single-tube detection and single-gene detection, and its sensitivity is achieved through a two-step method, that is, RPA amplification first, and on the basis of amplification, the amplification product needs to be added to the CrRNA / LwaCas13a reaction system to achieve. It is also inconvenient to operate and use

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