摘要
目的建立适合检测蚊虫携带黄病毒属病毒通用引物反转录半巢式PCR(RT-heminested-PCR)方法。方法根据GenBank黄病毒属病毒非结构蛋白基因(nonstructuralproteins gene)NS5序列,在保守区设计2对(3条)黄病毒属病毒通用引物,以日本脑炎病毒(Japanese encephalitis virus,JEV)cDNA、登革病毒(Dengue virus,DENV)Ⅰ～Ⅳ型cDNA为模板优化条件建立RT-heminested-PCR方法;以日本脑炎病毒减毒活疫苗(SA14-14-2strain)测定该方法的敏感度,并用于野外采集蚊虫检测。结果在50只淡色库蚊中RT-heminested-PCR方法检测减毒活疫苗JEV最低检出浓度为1×10-2 PFU/mL。用该方法检测云南省普洱市采集的54组(540只)蚊虫,扩增产物经测序确认9组含有乙型脑炎病毒﹑3组含有登革病毒II型﹑1组含有登革病毒I型﹑1组含未报道的黄病毒属病毒,该病毒与Quang Binh virus同源性最高。结论黄病毒属病毒通用引物RT-heminested-PCR方法适合于蚊虫种群黄病毒属病毒监测。其不但适应已知黄病毒属病毒的检测,而且具备检测新型黄病毒属病毒的潜能。
To establish a RT-heminested-PCR method for detecting flavivirus in Mosquitoes. We designed two pairs of universal primers targetting the conserved regions within nonstructural proteins gene （ NS5） of the Flavivirus genus. cDNA of JEV and DENV types I～IV were used as template to optimize the reaction conditions. The sensitivities of the assay was evaluated with attenuated vaccine strain of JEV （SA14-14 -2）. Furthermore, we validated the RT-heminested-PCR assay with field-caught mosquitoes grouped in pools. The threshold concentration was 1.0×10-2 PFU/mL for detecting JEV in pool of 50 mosquitoes. RT-hemi-nested PCR assay was used to detect Culex tritaeniorhynchus collected from Puer city of YunNan province in China. Amplified sequences were identified by sequencing. Of the 54 pools tested, 9 pools contained JEV, 3 pools contained DENV type II, 1 pool contained DENV type I, and 1 pool was positive for a previously undescribed flaviviral sequence that was most similar to Quang Binh virus. These data suggest that the RT-heminested-PCR using genus universal primers can be adopted for flavivirus surveillance in mosquito populations, and this approach has the potential to detect both previously recognized and novel viruses.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2013年第7期653-658,共6页
Chinese Journal of Zoonoses
基金
国家科技重大专项(2012ZX10004-220
2008ZX10004-011)~~
