Multiplex isothermal helicase-dependent amplification assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae
Multiplex isothermal helicase-dependent amplification assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae
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DOI：
10.1016/j.diagmicrobio.2011.08.021
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发表时间：
2011-12-01
影响因子：
2.9
通讯作者：
Nazarenko, Irina
中科院分区：
文献类型：
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作者：
Doseeva, Victoria;Forbes, Thomas;Nazarenko, Irina
嗜热解旋酶依赖性扩增（Thermophilic helicase dependent amplification，tHDA）是一种较新的等温核酸扩增技术，它利用解旋酶在恒温条件下解旋双链DNA。在这项研究中，4重tHDA检测沙眼衣原体（CT）和淋病奈瑟菌（NO）的开发和优化进行了描述。将tHDA与磁珠上的序列特异性样品制备和使用双标记探针的均匀终点荧光检测相结合。在存在内部对照的情况下，该4重tHDA测定法用于检测CT上的2个基因和NG上的多拷贝基因。该检测方法具有较高的分析灵敏度和特异性，可同时检测CT/NG，并与多种样品类型和培养基兼容。该分析中使用的等温反应条件和均相终点检测非常适合实验室自动化和高通量筛选应用以及即时检测。(C)2011 Elsevier Inc. All rights reserved.
Thermophilic helicase dependent amplification (tHDA), which employs helicase to unwind double-stranded DNA at constant temperature, is a relatively new isothermal nucleic acid amplification technology. In this study, the development and optimization of a 4-plex tHDA assay for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NO) are described. tHDA is combined with sequence-specific sample preparation on magnetic beads and homogeneous endpoint fluorescence detection using dual-labeled probes. This 4-plex tHDA assay was applied to the detection of 2 genes on CT and a multicopy gene on NG in the presence of an internal control. The assay showed high analytical sensitivity and specificity of simultaneous CT/NG detection and is compatible with a wide variety of sample types and media. The isothermal reaction conditions and homogeneous endpoint detection utilized in this assay are well suited for laboratory automation and high-throughput screening applications as well as for point-of-care testing. (C) 2011 Elsevier Inc. All rights reserved.