TaqMan荧光定量RT—PCR快速检测西尼罗病毒的研究

目的建立特异、快速、灵敏的TaqMan实时荧光定量聚合酶链反应（TaqMan—based Real—timePCR）用于西尼罗病毒（WNV）核酸检测，为WNV的感染诊断和流行病学监测奠定技术基础。方法根据文献公开发表的WNV核酸检测引物和TaqMan探针，优选最佳引物探针。应用DNAWorks2．4在线软件设计8条寡核苷酸片段，基于PCR合成包含引物探针扩增区域的230个核苷酸作为WNV模拟核酸。并对TaqMan-based Real—timePCR条件进行优化，验证该方法的特异性、灵敏度。结果该方法对WNV核酸检测有高度特异性，与流行性乙型脑炎、登革热1～4型等虫媒病毒均无交叉反应，对构建的DNA检测灵敏度达100拷贝／反应。结论TaqMan—based Real—timePCR是一种快速检测WNV特异、敏感的方法，适用于WNV的早期感染诊断和流行病学监测。

Study on quick detection of West Nile virus by TaqMan-based Real-time PCR

Objective To establish a TaqMan-based Real-time PCR assay for detection of West Nile virus. Methods The specific primers and probe selected from the references were synthesized in the conserved region of the CAP gene of West Nile virus, and the viral nucleic acid was synthesized by PCR-based gene synthesis method. The PCR condition was optimized to improve the sensitivity and specificity of the assay. Results The specificity of the assay was high and there were no cross reactions with Dengue virus and Japanese encephalitis virus. The sensitivity of the assay was 100 gene copies per cycle. Conclusion This TaqMan-based Real-time PCR assay was a quick, sensitive and specific tool for molecular diagnosis of West Nile virus.