东方马脑脊髓炎病毒TaqMan MGB荧光定量RT-PCR检测方法的建立  

Establishment of TaqMan MGB Real-time RT-PCR Assay for Detection of Eastern Equine Encephalitis Virus

作　　者：郑小龙[1] 王群[1] 张晓文[1] 孙明君[1] 朱来华[1] 

机构地区：[1]山东出入境检验检疫局,青岛266002

出　　处：《中国畜牧兽医》2015年第11期2850-2855,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家质检总局科技项目(2014IK240)

摘　　要：本试验通过RT-PCR从东方马脑脊髓炎病毒（EEEV）中扩增得到128bp的特异性保守序列,将其克隆到pMD20-T载体中,并进行体外转录,制备标准品cRNA。以10倍系列稀释的cRNA为模板,进行TaqMan MGB荧光定量RT-PCR扩增并制作标准曲线,建立了EEEV TaqMan MGB荧光定量RT-PCR的检测方法。进一步对建立的方法进行了特异性和敏感性试验。结果表明,建立的TaqMan MGB荧光定量RT-PCR最低可检测10拷贝/μL的cRNA;且与阴性对照及马鼻肺炎病毒（EHV-1）、马动脉炎病毒（EAV）、马流感病毒（EIV,H3N8）、西尼罗病毒（WNV）、西方马脑脊髓炎病毒（WEEV）和日本马脑炎病毒（JEV）均不发生交叉反应。所制作的标准曲线在1.0×10^1~1.0×10^6拷贝/μL浓度范围内有极好的线性关系,相关系数为0.998,标准曲线方程式为y=40-3.35logx;与常规RT-PCR相比,该方法更加快速,特异性和敏感性更高,灵敏度为常规RT-PCR方法的100倍。试验结果表明,建立了一种快速、高效、特异、敏感的EEEV TaqMan MGB荧光定量RT-PCR检测方法。The 128 bp specific and consensus sequence of Eastern equine encephalitis virus（EEEV）was amplified by RT-PCR and cloned into pMD20-T vector,and conducted in vitro transcription to prepare standard cRNA.10 fold serial diluted cRNA were used as standard templates for RT-PCR to quantify the genomic copy number of EEEV.We developed a TaqMan MGB Real-time RT-PCR to detect EEEV.Sensitivity assay result showed that the established TaqMan MGB Real-time RT-PCR could detect 10copies/μL cRNA.The specificity assay exhibited that negative control and the other equine pathogens（EHV-1,EAV,EIV H3N8,WNV,WEEV and JEV）could not be detected.A good linear correlation was demonstrated in the standard curve for TaqMan MGB Real-time RT-PCR within the range of 1.0×10^1 to 1.0×10^6 copies/μL with a correlation coefficient of 0.998 and a standard curve of y=40-3.35 logx.The results demonstrated that TaqMan MGB Real-time RT-PCR was 100 fold more sensitive than conventional RT-PCR.The results suggested that the convenient,specific,high performance and sensitive method of TaqMan MGB Real-time RT-PCR for the detection of EEEV was successfully established in this study.

关 键 词：东方马脑脊髓炎病毒 TAQMAN MGB 荧光定量RT-PCR 

分 类 号：Q78[生物学—分子生物学]