猪戊型肝炎病毒SYBR Green Ⅱ荧光定量RT-PCR检测方法的建立与应用  

Development and application of a SYBR Green Ⅱ real-time RT-PCR for detection of swine hepatitis E virus

作　　者：李幽幽 龚双燕[1] 李小璟 毛汐语 邓益超 朱玲[1,2] 徐志文[1,2] 

机构地区：[1]四川农业大学动物医学院,成都611134 [2]四川农业大学动物疫病与人类健康四川省重点实验室,成都611134

出　　处：《中国人兽共患病学报》2017年第11期1002-1006,1017,共6页Chinese Journal of Zoonoses

基　　金：四川省科技支撑计划(No.2014NZ0043);国家"十二五"科技支撑计划(No.2015BAD12B04-2.3)联合资助~~

摘　　要：目的建立一种快速检测猪戊型肝炎病毒(HEV)的方法。方法针对HEVORF2基因保守序列设计1对引物,基于SYBR GreenⅡ染料,建立了检测HEV的实时荧光定量RT-PCR方法。结果该方法只能对猪戊型肝炎病毒检测到荧光,对猪繁殖与呼吸综合征病毒、猪瘟病毒、猪流行性腹泻病毒、猪嵴病毒、猪传染性胃肠炎病毒、猪轮状病毒均不能检测到荧光信号,特异性好;检测HEV时,在4.10×10~2~4.10×10~8拷贝/μL内具有良好的线性关系,灵敏度可以达到1.00×10~2拷贝/μL,扩增相关系数为0.996,扩增产物的熔解温度为84.0℃±0.1℃,该检测方法组内变异系数为0.83%~0.94%,组间变异系数为0.83%~0.94%,重复性较好。利用该方法对临床61份来自四川各地的猪肠内容物进行检测,检出9份阳性,且与常规PCR检测方法的阳性符合率为100%。结论建立的针对HEV的荧光定量RT-PCR有效可行,对HEV的临床检测和进一步研究奠定了基础。In order to establish a real-time RT-PCR based on SYBR Green Ⅱfor detection of hepatitis E virus(HEV),a pair of special primers was designed according to the conserved sequences of ORF2 in GenBank.Result showed that the standard curve of established SYBR Green Ⅱreal-time RT-PCR had a wide dynamic range from 4.10×10~2-4.10×10~8 copies/μL with a linear correlation(r^2)of 0.996.The sensitivity could reach 1.00×10~2 copies/μL.The melting curve analysis using SYBR GreenⅡ dye showed one specific peak with a melting temperature(Tm)of 84.0℃±0.1℃.No amplification was detected from the RNA samples of porcine reproductive and respiratory syndrome virus,classial swine fever virus,transmissible gastroenteritis virus,porcine bocavirus,porcine epidemic dearrhoea virus porcine kobuvirus and porcine rotavirus by this PCR,respectively.Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.83%-0.94% and inter-assay of 0.83%-0.94%.Further detection of 61 specimens showed that 9 of them were HEV positive,and the results of the quantitative RT-PCR were the same as that of the conventional RT-PCR.In conclusion,the real-time quantitative RT-PCR for HEV is feasible,the real-time RT-PCR established in this study will be useful for earlier rapid laboratory diagnosis and pathogenesis of HEV.

关 键 词：猪戊型肝炎病毒 SYBR GreenⅡ 荧光定量RT-PCR 

分 类 号：R373.2[医药卫生—病原生物学]