摘要
目的建立针对云南地区流行的基因Ⅲ型乙型脑炎病毒株Real-time PCR检测方法。方法根据云南地区分离株乙型脑炎病毒株(Yunnan0901)基因组核苷酸序列设计特异性引物,构建pMD18-T-JEV-E质粒,以此为模板进行SYBR GreenⅠReal-time PCR扩增并制作标准曲线,摸索最佳反应体系条件,建立乙型脑炎病毒荧光定量PCR检测方法,并与普通RT-PCR方法相比较。结果建立的SYBR GreenⅠReal-time PCR标准曲线呈现良好的重复性和特异性,与模板浓度呈现良好的线性关系。与RT-PCR方法相比,SYBR GreenⅠReal-time PCR的灵敏度高10倍,而且不与猪瘟病毒、猪蓝耳病毒、猪圆环病毒、基孔肯雅病毒、辛德毕斯病毒核酸发生非特异性扩增,具有良好的特异性。用该方法检测云南部分地区的蚊虫样品,其中库蚊、按蚊乙型脑炎病毒阳性率分别为17.4%和30.7%,且主要为基因型Ⅲ型。结论本实验建立的检测基因Ⅲ型乙型脑炎病毒的SYBR GreenⅠReal-time PCR方法具有特异、敏感、快速、定量、重复性好等特点,可应用于乙型脑炎疫情的监测。
Objective To develop a SYBR Green Ⅰ real-time quantitative PCR assay for detection of Japanese encephalitis virus（JEV） genotype Ⅲ in the Yunnan region.Methods Primers were designed according to the specific nucleotide sequence of the JEV.The recombinant plasmid pMD18-T-JEV-E was constructed and served as a template to prepare the standard curve for SYBR Green Ⅰ real-time PCR.Determination of optimal PCR conditions helped to establish SYBR Green Ⅰ real-time PCR for JEV genotype Ⅲ and its sensitivity was compared to that of traditional RT-PCR.Results The results demonstrated that the standard curve established with the recombinant plasmid had a good linear relationship between threshold cycle and template concentration with good reproducibility and specificity.The sensitivity of real-time PCR was ten times greater than that of traditional RT-PCR.The specificity assay revealed no amplification of other porcine pathogens such as PCV-2,PRRSV,CSFV,CHIKV,and SINV.Culex and Anopheles mosquitoes from Yunnan Province tested positive for JEV at a rate of 17.4% and 30.7%,respectively,and JEV was genotype Ⅲ.Conclusion Results indicated that SYBR Green Ⅰ real-time PCR was a rapid,specific,and sensitive method of detecting JEV.This technique could be used to monitor the prevalence of JEV.
出处
《中国病原生物学杂志》
CSCD
北大核心
2012年第9期644-648,656,共6页
Journal of Pathogen Biology
基金
公益性行业(农业)科研项目(No.201203082)
