tetano
Editor, Senior Moderator
J Med Virol
. 2020 Jun 12.
doi: 10.1002/jmv.26171. Online ahead of print.
A Simple Method for Detection of a Novel Coronavirus (SARS-CoV-2) Using One-step RT-PCR Followed by Restriction Fragment Length Polymorphism
Ho Anh Son[SUP] 1 [/SUP], Dinh Thi Thu Hang[SUP] 1 [/SUP], Nghiem Duc Thuan[SUP] 2 [/SUP], Le Thi Bao Quyen[SUP] 1 3 [/SUP], Luong Thi Hoai Thuong[SUP] 1 3 [/SUP], Vu Thi Nga[SUP] 1 3 [/SUP], Le Bach Quang[SUP] 4 [/SUP], Trinh Thanh ****[SUP] 4 [/SUP], Nguyen Thai Son[SUP] 5 [/SUP], Nguyen Tung Linh[SUP] 6 [/SUP], Le Van Nam[SUP] 7 [/SUP], Nguyen Van Ba[SUP] 8 [/SUP], Tran Viet Tien[SUP] 8 [/SUP], Do Quyet[SUP] 9 [/SUP], Hoang Van Luong[SUP] 1 [/SUP], Hoang Xuan Su[SUP] 1 [/SUP]
Affiliations
Abstract
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.
Keywords: Coronavirus Disease: SARS-CoV-2; RT-PCR-RFLP; SARS-CoV.
. 2020 Jun 12.
doi: 10.1002/jmv.26171. Online ahead of print.
A Simple Method for Detection of a Novel Coronavirus (SARS-CoV-2) Using One-step RT-PCR Followed by Restriction Fragment Length Polymorphism
Ho Anh Son[SUP] 1 [/SUP], Dinh Thi Thu Hang[SUP] 1 [/SUP], Nghiem Duc Thuan[SUP] 2 [/SUP], Le Thi Bao Quyen[SUP] 1 3 [/SUP], Luong Thi Hoai Thuong[SUP] 1 3 [/SUP], Vu Thi Nga[SUP] 1 3 [/SUP], Le Bach Quang[SUP] 4 [/SUP], Trinh Thanh ****[SUP] 4 [/SUP], Nguyen Thai Son[SUP] 5 [/SUP], Nguyen Tung Linh[SUP] 6 [/SUP], Le Van Nam[SUP] 7 [/SUP], Nguyen Van Ba[SUP] 8 [/SUP], Tran Viet Tien[SUP] 8 [/SUP], Do Quyet[SUP] 9 [/SUP], Hoang Van Luong[SUP] 1 [/SUP], Hoang Xuan Su[SUP] 1 [/SUP]
Affiliations
- PMID: 32530490
- DOI: 10.1002/jmv.26171
Abstract
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.
Keywords: Coronavirus Disease: SARS-CoV-2; RT-PCR-RFLP; SARS-CoV.