摘要
本试验旨在建立同步共检猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(RV)的多重RT-PCR检测方法。参照PEDV的M基因(JQ023161)、TGEV的N基因(JX013850),RV的VP7基因(AY707788),利用Primer Premier 5.0软件设计筛选能特异性扩增PEDV、TGEV、RV相应基因的引物。并对引物浓度、退火温度等扩增条件进行优化,建立了一种能同时共检PEDV、TGEV和RV的多重RT-PCR方法。该方法对PEDV、TGEV和RV均能分别扩增出大小为347、779和510bp的预期片段;该方法灵敏度高,最低可以检测约1.5×105拷贝的阳性RNA模板;该检测方法特异性强,TGEV、PEDV、RV三种病毒核酸检测体系内无交叉反应,对猪瘟病毒、猪繁殖与呼吸综合征病毒、日本脑炎病毒和伪狂犬病病毒的RNA或DNA检测均为阴性。应用该方法检测135份临床样品,TGEV、PEDV和RV的阳性检出率分别为2.96%、82.2%和2.22%,与常规单一RT-PCR的检测结果进行比较分析,符合率均为100%。上述研究结果表明该方法是一种简便、快速、同步共检PEDV、TGEV和RV的有效方法。
The experiment was designed to establish a multiplex RT-PCR that could simultaneously detect porcine epidemic diarrhea virus（PEDV）,transmissible gastroenteritis virus of swine（TGEV）and porcine rotavirus（RV）.Three pairs of special primers were designed by Primer Premier 5.0,targeting the M gene（JQ023161）of PEDV,N gene（JX013850）of TGEV and VP7gene（AY707788）of RV,respectively.Using the three pairs of primers,the primer concentration,annealing temperature and other amplification conditions were optimized,and a multiplex RT-PCR was successfully established.The method could amplify the expected fragment of PEDV,TGEV and RV,whose size were 347 bp,779bp and 510 bp,respectively.The sensitivity test showed that the detection method held a high sensitivity,and a minimum copy RNA template of 1.5×10^5 could be detected.In the specificity test,there were no cross reactions found in the detection,and all the detection of DNA or RNA of classical swine fever virus,porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus and pseudorabies virus were negative,showing agood specificity.A total of 135 clinic samples of sick pigs were detected by the multiplex RT-PCR and the positive rate was 2.96%（4/135）for TGEV,82.2%（111/135）for PEDV and 2.22%（3/135）for RV.Which were consistent with the result using the routine single RT-PCR,and that the coincidence rate was 100%.All this indicated that the multiplex RT-PCR with high sensitivity and specificity is a new rapid effective method for the simultaneous detection of PEDV,TGEV and RV.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2015年第9期881-887,共7页
Chinese Veterinary Science
基金
国家级大学生创新创业训练计划项目(201310626003)
