Universal primer sequence for detecting human coronavirus infection and detection method

Abstract

The invention designs a pair of degenerate primers hCoV-For and hCoV-Rev applied to universal qualitative detection of human coronavirus in a genomic polymerase gene coding region (1b region) by virtue of comparison analysis of six human coronavirus complete genomic nucleotide sequences logged in GenBank. An upstream primer sequence hCoV-For is as follows: ATGGGWTGGGAYTAYCCHAARTGTG; and downstream primer sequence hCoV-Rev is as follows: TGYTGKGARCARAAYTCRTGWGG; the size of an amplification target fragment is 605bp; RNA of positive specimens of HCoV-229E, HCoV-OC43, HCoC-NL63 and HCoV-HKU1 are respectively extracted for testing the universal primer by One-step RT-PCR. Results show that four human coronavirus RNAs are used as templates, and hCoV-For and hCoV-Rev are used as the universal primers for carrying out One-step RT-PCR reaction, a single target strip with the size about 606p can be amplified; by carrying out sequencing validation of PCR products, results show that the amplification sequence is right without cross reaction. The universal primer sequence can be applied to quick and qualitative detection of nucleic acid of human coronavirus infection.

Description

technical field [0001] The invention belongs to the fields of molecular biology and virology, and more specifically relates to a novel one-step RT-PCR reaction using a pair of degenerate primers to achieve the purpose of qualitative detection of human coronavirus infection. Background technique [0002] Coronavirus (Coronavirus) belongs to the order Nidovirals (order Nidovirals), Coronaviridae (family Coronavirade), and members of the genus Coronavirus (genus Coronavirus) in virological classification. Between 26 and 32 kb, it is the virus with the largest genome among known RNA viruses. The infection of coronaviruses in nature is generally very widespread, and common mammals such as dogs, cats, mice, pigs, cattle, and poultry are susceptible. In recent years, various types of coronaviruses have been isolated from beluga whales, camels, and especially bats. Based on the phylogenetic analysis of the nucleic acid sequence of coronaviruses, the International Committee on Viro...

AI Technical Summary

Problems solved by technology

These methods usually use specific primer needles and probes to perform a single detection of a specific type of human coronavirus, which is a blind screening test, which is not only time-consuming and laborious, but also the detection reagents are expensive and easy to cause pollution

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