Development of isothermal TaqMan assays for detection of biothreat organisms. (November 2008)

• Record Type: Journal Article
• Authors: Tong, Yanhong; Tang, Wen; Kim, Hyun-Jin; Pan, Xiaojing; Ranalli, Tamara A.; Kong, Huimin
• Is Part Of: Biotechniques. Volume 45:Number 5(2008)
• Page Start: 543
• Page End: 557
• Publication Date: 2008-11
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/000112959
• Languages: English
• ISSNs: 0736-6205

Abstract

TaqMan probe (dual-labeled DNA probe)–based real-time detection, one of the most sensitive and specific fluorescent detection methods, has been widely utilized in conjunction with polymerase chain reaction (PCR). Helicase-dependent amplification (HDA) is an isothermal amplification technology that has a similar reaction scheme to PCR, but replaces thermocycling with a helicase capable of unwinding a DNA duplex. Here we describe a novel isothermal real-time detection method (HDA-TaqMan) that combines the advantages of both HDA and a TaqMan assay. In this assay, the reactions of DNA unwinding, primer annealing, polymerization, probe hybridization, and subsequent hydrolysis by the polymerase are coordinated and synchronized to perform at a single temperature. It not only provides a useful tool for real-time detection of HDA, but also provides an isothermal format for the TaqMan system. With this platform, we have successfully developed rapid real-time isothermal assays for biodefense targets that include Vibrio cholerae and Bacillus anthracis .