A simple method for detection of a novel coronavirus (SARS‐CoV‐2) using one‐step RT‐PCR followed by restriction fragment length polymorphism

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 est...

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Veröffentlicht in:Journal of medical virology 2020-11, Vol.92 (11), p.2839-2846
Hauptverfasser: 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
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Sprache:eng
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Zusammenfassung: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 EcoRI, Tsp45I, and AluI 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 Tsp45I 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 EcoRI producing two fragments of 76 and 320 bp, whereas the amplicon of SARS‐CoV‐2 was undigested by Tsp45I help to distinguish clearly SARS‐CoV‐2 from SARS‐CoV on gel electrophoresis. In addition, AluI 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. Highlights We developed a simple method for molecular detection of SARS‐CoV‐2 in differentiating with SARS‐CoV The assay showed comparable analytical, clinical performance and overall agreement to other reference assays on clinical samples
ISSN:0146-6615
1096-9071
DOI:10.1002/jmv.26171