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A simple method for detection of a novel coronavirus (SARS‐CoV‐2) using one‐step RT‐PCR followed by restriction fragment length polymorphism
Author(s) -
Son Ho Anh,
Hang Dinh Thi Thu,
Thuan Nghiem Duc,
Quyen Le Thi Bao,
Thuong Luong Thi Hoai,
Nga Vu Thi,
Quang Le Bach,
Hung Trinh Thanh,
Son Nguyen Thai,
Linh Nguyen Tung,
Nam Le Van,
Van Ba Nguyen,
Tien Tran Viet,
Quyet Do,
Van Luong Hoang,
Su Hoang Xuan
Publication year - 2020
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.26171
Subject(s) - amplicon , biology , ecori , virology , restriction fragment length polymorphism , polymerase chain reaction , restriction enzyme , coronavirus , gene , microbiology and biotechnology , restriction fragment , genetics , covid-19 , medicine , infectious disease (medical specialty) , disease , pathology
A novel coronavirus associated with acute respiratory disease (named SARS‐CoV‐2) is recently identified in Wuhan city, China, spread rapidly worldwide. Early identification of this novel coronavirus by molecular tools is critical for surveillance and control of the epidemic outbreak. We aimed to establish a simple method for the detection of SARS‐CoV‐2 in differentiating with SARS‐CoV. Primers of our in‐house reverse transcription polymerase chain reaction (RT‐PCR) assays were designed to target conserved regions of the RdRP gene and E gene, selected restriction enzymes Eco RI, Tsp 45I, and Alu I to distinguish between SARS‐CoV‐2 and SARS‐CoV. In this report, a 396‐bp fragment of the RdRp gene and 345‐bp fragment of the E gene were amplified by one‐step RT‐PCR. Enzyme Tsp 45I cuts the RdRP‐amplified product of SARS‐CoV‐2 generating three fragments of 45, 154, and 197 bp, but it did not cut the amplicon of SARS‐CoV. In contrast, the amplified product of SARS‐CoV was digested with Eco RI producing two fragments of 76 and 320 bp, whereas the amplicon of SARS‐CoV‐2 was undigested by Tsp 45I help to distinguish clearly SARS‐CoV‐2 from SARS‐CoV on gel electrophoresis. In addition, Alu I cut the amplicon of the E gene of SARS‐CoV‐2 generating two fragments of 248 and 97 bp without cutting to SARS‐CoV. The accuracy of the assay was confirmed by sequencing and phylogenetic analysis. When evaluated on clinical samples showed a high sensitivity of 95%, specificity of our assay was 100% and clinical performance for detection of SARS‐CoV‐2 in comparison with other reference assays. In conclusion, in the present study, we successfully developed a simple method for molecular detection of SARS‐CoV‐2 in differentiating with SARS‐CoV.