PEDV和TGEV双重TaqMan荧光定量一步法RT-PCR检测方法的建立及应用

摘要

In the present study,one step duplex TaqMan fluorescent real-time RT-PCR assay of PEDV and TGEV was developed to detect and differentiate Porcine epidemic diarrhea virus,(PEDV) and Transmissible gastroenteritis virus,(TGEV).The specific primers and TaqMan fluorescent probes of these viruses were designed based on the bio-information analysis and then the amplification condition and the concentration of each virus were optimized.The result showed that the detection limits were 101 copies for both PEDV and TGEV and there was no cross reaction with other common viruses.Subsequently,one step Duplex TaqMan Real-time PCR assay was assembled into the PEDV and TGEV Detection Kit.The inter and intra-assay trials demonstrated that the variations were less than 0.58％ and 3.7％,respectively,indicating its good reproducibility.Total 97 samples were examined using this kit and the positive rate of PEDV and TGEV was 14.93％ higher than the current PCR method.These results demonstrated that this assay was a rapid,specific and sensitive method for detection of PEDV and TGEV.In conclusion,the method established in the present study can be used for rapid detection and epidemiology study of PEDV and TGEV.%本文通过对猪腹泻病毒(Porcine epidemic diarrhea virus,PEDV)和猪传染性胃肠炎病毒(Transmissible gastroenteritisvirus,TGEV)基因组生物信息学分析,分别在两种病毒基因保守区设计特异性探针和Real-Time PCR引物,并建立了检测PEDV和TGEV的双重实时荧光定量RT-PCR方法.结果表明,本研究建立的方法对其他常见病毒无交叉检出,具有较强的特异性:应用本方法对PEDV和TGEV检测灵敏度均可达101个拷贝.应用本法组装PEDV和TGEV双重实时荧光定量PCR试剂盒,该试剂盒批次内、批次间的变异系数CV(coefficient of variation,Cv)分别低于0.58％和3.7％.应用该试剂盒对97份病例进行检测,阳性率提高了14.93％.本研究建立的一步法荧光定量RT-PCR方法具有快速、特异性好、灵敏度高、定量且重复性和稳定性好等优点,在PEDV和TGEV感染的快速鉴别诊断,以及流行病学调查方面都有广阔的应用前景.