Development of isothermal TaqMan assays for detection of biothreat organisms
Development of isothermal TaqMan assays for detection of biothreat organisms
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DOI：
10.2144/000112959
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发表时间：
2008-11-01
期刊：
影响因子：
2.7
通讯作者：
Kong, Huimin
中科院分区：
文献类型：
--
作者：
Tong, Yanhong;Tang, Wen;Kong, Huimin
基于TaqMan探针（双标记DNA探针）的实时检测是最灵敏、最特异的荧光检测方法之一，与聚合酶链反应（PCR）结合使用已得到广泛应用。解旋酶依赖性扩增（HDA）是一种等温扩增技术，其具有与PCR类似的反应方案，但用能够解旋DNA双链体的解旋酶代替热循环。在这里，我们描述了一种新的等温实时检测方法（HDA-TaqMan），结合了HDA和TaqMan检测的优点。在该测定中，DNA解旋、引物退火、聚合、探针杂交和随后的聚合酶水解的反应在单一温度下与穿孔素协调和同步。它不仅为HDA的实时检测提供了有用的工具，而且为TaqMan系统提供了等温格式。利用这个平台，我们已经成功地开发了快速实时等温检测生物防御目标，包括霍乱弧菌和炭疽杆菌。
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 perforin 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.