Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes
Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes
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DOI：
10.1128/jcm.00842-06
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发表时间：
2007-02-01
影响因子：
9.4
通讯作者：
Chang, Gwong-Jen J.
中科院分区：
文献类型：
--
作者：
Chao, Day-Yu;Davis, Brent S.;Chang, Gwong-Jen J.
建立了一种多重实时逆转录PCR方法，用于从实验室饲养的病毒感染蚊池中快速检测和鉴定8种医学上重要的黄病毒。所使用的方法涉及黄热病病毒（YFV）、日本脑炎病毒（JEV）、西尼罗河病毒（WNV）、圣路易斯脑炎病毒（SLEV）和登革病毒（DENV）血清型1至4（分别为DENV-1至DENV-4）的基因特异性扩增，方法是使用位于非结构蛋白5的RNA依赖性RNA聚合酶结构域的哈维病毒共有扩增物。病毒特异性扩增子通过四种新表征的TaqMan荧光探针（特异于YFV、JEV、V* NV和SLEV的探针）和四种先前公开的特异于DENV-1至DENV-4（L. J. Chien，T. L. Liao，P. Y. Shu，J.H. Huang，黄氏拟谷盗D. J. Gubler和G. J. Chang，J. Clin. Microbiol。44：1295-1304，2006）。该方法的特异性为100%，灵敏度为：YFV至少3.5 PFU/ml，JEV至少2.0 PFU/ml，WNV至少10.0 PFU/ml，SLEV至少10.0 PFU/ml。此外，我们还开发了一种体外转录系统，为这八种病毒中的每一种生成RNA酶抗性RNA模板。这些模板可以作为RNA拷贝数对照和/或作为RNA掺入的蚊子库的外部对照掺入测定中以用于质量保证目的。尽管还需要对现场收集的蚊子进行进一步研究，但将该检测方法纳入蚊子监测中可用作检测医学上重要的黄病毒的预警系统，特别是当怀疑同一地区多种病毒共循环时。
A multiplex real-time reverse transcriptase PCR has been developed for the rapid detection and identification of eight medically important flaviviruses from laboratory-reared, virus-infected mosquito pools. The method used involves the gene-specific amplification of yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus (DENV) serotypes 1 to 4 (DENV-1 to DENV-4, respectively) by use of the Havivirus consensus amplimers located at the RNA-dependent RNA polymerase domain of nonstructural protein 5. Virus-specific amplicons were detected by four newly characterized TaqMan fluorogenic probes (probes specific for YFV, JEV, V*`NV, and SLEV) and four previously published probes specific for DENV-1 to -4 (L. J. Chien, T. L. Liao, P. Y. Shu, J. H. Huang, D. J. Gubler, and G. J. Chang, J. Clin. Microbiol. 44:1295-1304, 2006). This assay had a specificity of 100% and various sensitivities of at least 3.5 PFU/ml for YFV, 2.0 PFU/ml for JEV, 10.0 PFU/ml for WNV, and 10.0 PFU/ml for SLEV. Additionally, we have developed an in vitro transcription system to generate RNase-resistant RNA templates for each of these eight viruses. These templates can be incorporated into the assay as RNA copy number controls and/or as external controls for RNA-spiked mosquito pools for quality assurance purposes. Although further study with mosquitoes collected in the field is needed, the incorporation of this assay into mosquito surveillance could be used as an early-warning system for the detection of medically important flaviviruses, particularly when the cocirculation of multiple viruses in the same region is suspected.