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A high-specificity flap probe-based isothermal nucleic acid amplification method based on recombinant FEN1-Bst DNA polymerase.
Ye, Xin; Wang, Ning; Li, Yang; Fang, Xueen; Kong, Jilie.
  • Ye X; Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, PR China.
  • Wang N; State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
  • Li Y; Shanghai Suxin Biotechnology Co. Ltd., Shanghai, 201318, PR China.
  • Fang X; Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, PR China. Electronic address: fxech@fudan.edu.cn.
  • Kong J; Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, PR China. Electronic address: jlkong@fudan.edu.cn.
Biosens Bioelectron ; 192: 113503, 2021 Nov 15.
Article in English | MEDLINE | ID: covidwho-1309167
ABSTRACT
The COVID-19 pandemic has unfortunately demonstrated how easily infectious diseases can spread and harm human life and society. As of writing, pandemic has now been on-going for more than one year. There is an urgent need for new nucleic acid-based methods that can be used to diagnose pathogens early, quickly, and accurately to effectively impede the spread of infections and gain control of epidemics. We developed a flap probe-based isothermal nucleic acid amplification method that is triggered by recombinant FEN1-Bst DNA polymerase, which-through enzymatic engineering-has both DNA synthesis, strand displacement and cleavage functions. This novel method offers a simpler and more specific probe-primer pair than those of other isothermal amplifications. We tested the method's ability to detect SARS-CoV-2 (both ORF1ab and N genes), rotavirus, and Chlamydia trachomatis. The limits of detection were 10 copies/µL for rotavirus, C. trachomatis, and SARS-CoV-2 N gene, and 100 copies/µL for SARS-CoV-2 ORF1ab gene. There were no cross-reactions among 11 other common pathogens with characteristics similar to those of the test target, and the method showed 100% sensitivity and 100% specificity in clinical comparisons with RT-PCR testing. In addition to real-time detection, the endpoint could be displayed under a transilluminator, which is a convenient reporting method for point-of-care test settings. Therefore, this novel nucleic acid senor has great potential for use in clinical diagnostics, epidemic prevention, and epidemic control.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Biosensing Techniques / Chlamydia trachomatis / Rotavirus / SARS-CoV-2 Type of study: Diagnostic study / Prognostic study / Randomized controlled trials Limits: Humans Language: English Journal: Biosens Bioelectron Journal subject: Biotechnology Year: 2021 Document Type: Article

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Biosensing Techniques / Chlamydia trachomatis / Rotavirus / SARS-CoV-2 Type of study: Diagnostic study / Prognostic study / Randomized controlled trials Limits: Humans Language: English Journal: Biosens Bioelectron Journal subject: Biotechnology Year: 2021 Document Type: Article