A Simple Method for Detection of a Novel Coronavirus (SARS‐CoV‐2) using One‐step RT‐PCR followed by Restriction Fragment Length Polymorphism

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

BACKGROUND: A novel coronavirus associated with acute respiratory disease (named SARS‐CoV‐2) is recently identified in Wuhan city, China, spread rapidly worldwide. An early identification of this novel coronavirus by molecular tools is critical for surveillance and control of the epidemic outbreak. OBJECTIVES: We aimed to establish a simple method for detection of SARS‐CoV‐2 in differentiating with SARS‐CoV. STUDY DESIGN: Primers of our in‐house 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. RESULTS AND DISCUSSIONS: 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 3 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 2 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 2 fragments of 248 and 97 bp without cutting to SARS‐CoV. Accuracy of 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, the present study, we successfully developed a simple method for molecular detection of SARS‐CoV‐2 in differentiating with SARS‐CoV. This article is protected by copyright. All rights reserved.