猪环曲病毒实时荧光定量RT-PCR检测方法的建立与应用  

Establishment and application of a real-time RT-PCR method based on SYBR Green Ⅱ for detection of porcine torovirus

作　　者：周璐[1] 周远成[1] 魏浩澈[1] 付梦瑾[1] 刘骁[1] 季洪伟[1] 朱玲[1,2] 徐志文[1,2] 郭万柱[1,2] 

机构地区：[1]四川农业大学动物生物技术中心,四川雅安625014 [2]四川农业大学动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国兽医科学》2013年第9期952-957,共6页Chinese Veterinary Science

基　　金：四川省科技支撑计划项目(2012NZ0001);四川农业大学优秀硕士学位论文培育基金项目

摘　　要：根据GenBank中猪环曲病毒（porcine torovirus,PToV）N基因的保守序列,设计合成1对针对PToV的特异性引物,建立了基于SYBR GreenⅡ检测PToV的实时荧光定量RT-PCR方法。结果显示,该方法能很好地将PToV与猪伪狂犬病病毒、猪嵴病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、A群猪轮状病毒、猪瘟病毒、沙门菌和大肠杆菌区分开来,特异性强。检测PToV的N基因在8.65×101~8.65×108拷贝/μL范围内有很好的线性关系,其扩增相关系数为0.998 9,扩增产物的熔解曲线分析只有1个单特异峰。所建立的实时荧光定量RT-PCR方法组内变异系数为0.29%~1.30%,组间变异系数为0.45%~1.80%,重复性较好。利用该方法对采集自四川部分地区的43份临床样品进行检测,实时荧光定量RTPCR检出13份阳性样品,与常规PCR检测方法的阳性符合率为100%。结果表明建立的实时荧光定量RT-PCR方法特异性较强、灵敏度高,可用于该病的临床检测和流行病学调查。A pair of specific primers was designed according to the conserved sequences of the N gene of porcine torovirus in GenBank and a real-time reverse transcription polymerase chain reaction（RRT-PCR） based on SYBR Green 11 was developed for quantization of porcine torovirus. The result indicated that the standard curve generated a wide dynamic range of 8.65 × 10^1 to 8.65 ×10^8 copies/μL with a linear correla- tion（r2） of 0. 998 9. The melting curve analysis using SYBR Green 11 dye showed one specific peaked with no primer-dimer peak. No amplification was detected from other DNA samples by this method, such as por- cine pseudorabies virus, porcine kobuvirus, transmissible gastroenteritis virus of swine, porcine epidemic diarrhea virus, porcine rotavirus, classical swine fever virus, Salmonella, and Escherichia coll. Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.29% to 1.30% and inter-assay of 0.45% to 1.80%. Further detection of 43 clinical specimens showed that 13 of them were porcine torovirus positive by the real-time RT-PCR,and the results of the quantitative RT-PCR were the same as that of conventional RT-PCR. The results proved that the RRT-PCR was specific andsensitive to detect porcine torovirus, which can support the clinic detection and molecular epidemiological investigation.

关 键 词：猪环曲病毒 SYBR GreenⅡ 实时荧光定量RT-PCR 

分 类 号：S852.659.6[农业科学—基础兽医学]