Tong Yanhong, Tang Wen, Kim Hyun-Jin, Pan Xiaojing, Ranalli Tamara, Kong Huimin
BioHelix Corporation, Beverly, MA 01915, USA.
Biotechniques. 2008 Nov;45(5):543-57. doi: 10.2144/000112959.
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.
基于TaqMan探针（双标记DNA探针）的实时检测是最灵敏和特异的荧光检测方法之一，已与聚合酶链反应（PCR）一起被广泛应用。解旋酶依赖性扩增（HDA）是一种等温扩增技术，其反应方案与PCR相似，但用能够解开DNA双链的解旋酶替代了热循环。在此，我们描述了一种新型等温实时检测方法（HDA-TaqMan），它结合了HDA和TaqMan检测的优点。在该检测中，DNA解旋、引物退火、聚合、探针杂交以及随后的聚合酶水解反应在单一温度下协同且同步进行。它不仅为HDA的实时检测提供了一种有用的工具，也为TaqMan系统提供了一种等温形式。利用这个平台，我们已成功开发出针对包括霍乱弧菌和炭疽芽孢杆菌在内的生物防御靶点的快速实时等温检测方法。
