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目的建立用于检测黄病毒属的TaqMan探针荧光定量RT-PCR方法。方法从GenBank中检索黄病毒属代表株的全基因序列,通过DNAstar进行序列比对和blast进行保守序列搜索和分析,用Beacon Designer 7.0软件进行引物和TaqMan探针设计,以乙脑毒株MB090509 RNA为模板,优化反应条件并应用于现场蚊虫标本检测。结果确定黄病毒属TaqMan探针荧光定量RT-PCR反应条件为:45℃15 min,95℃10 min,95℃10 s→54℃45 s,40次循环,引物和探针终浓度为200 nmol/L。灵敏度为常规RT-PCR方法 100倍,检出限2.9×10-3噬斑形成单位(PFU)/mL,与甲病毒属、东南亚十二节段RNA病毒属、布尼亚病毒属均无交叉反应。从161份现场蚊虫标本中检测出11份阳性标本,经病毒分离、测序均被证实为乙脑病毒。结论本研究建立的黄病毒属TaqMan探针荧光定量RT-PCR方法具有广泛应用价值。
Objective To establish a real-time TaqMan probe real-time RT-PCR method for the detection of flavivirus. Methods Genomic DNA sequences of flavivirus representative strains were retrieved from GenBank. The sequences were searched and analyzed by DNAstar and blast. The primers and TaqMan probes were designed by Beacon Designer 7.0. As a template to optimize the reaction conditions and applied to the detection of mosquito specimens on the spot. Results The quantitative RT-PCR reaction conditions of flavivirus TaqMan probe were as follows: 45 ℃ for 15 min, 95 ℃ for 10 min, 95 ℃ for 10 s → 54 ℃ for 45 s, 40 cycles, primer and probe final concentration was 200 nmol / L. The sensitivity was 100-fold with the conventional RT-PCR method and the detection limit was 2.9 × 10-3 PFU / mL. There was no crossover with the alphavirus, twelve RNAi and Bunia viruses in Southeast Asia reaction. Eleven positive samples were detected from 161 mosquitoes in the field. After virus isolation and sequencing, they were all confirmed as JE virus. Conclusion The fluorescence quantitative RT-PCR method of TaqMan probe of flavivirus in this study has a wide range of applications.