CSFV与PRRSV双重RT-PCR检测方法的建立及应用  

Establishment and application of duplex RT-PCR method of CSFV and PRRSV

作　　者：杨伟 单春兰 李林 高洪 严玉霖 赵汝 敖平星 高利波 刘超英[2] 富国文 高斌[3] 景麟稀 茶金龙 王浩 YANG Wei;SHAN Chunlan;LI Lin;GAO Hong;YAN Yulin;ZHAO Ru;AO Pingxing;GAO Libo;LIU Chaoying;FU Guowen;GAO Bin;JING Linxi;CHA Jinlong;Wang Hao(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China;College of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China;College of Food Science and Technology,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学动物医学院,云南昆明650201 [2]云南农业大学动物科学技术学院,云南昆明650201 [3]云南农业大学食品科学技术学院,云南昆明650201

出　　处：《畜牧与兽医》2019年第10期71-75,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金资助项目(31660704,31260594);云南省现代农业生猪产业技术体系建设(云财农[2009]171号);云南省高校科技创新团队支持计划资助项目(云教科[2011]14号)

摘　　要：为建立能同时检测猪瘟病毒(CSFV)和猪繁殖与呼吸综合征病毒(PRRSV)的RT-PCR方法,根据CSFV和PRRSV的保守区域各设计1对特异性引物,预期扩增片段大小分别为356和546 bp,通过对反应条件的优化及特异性、灵敏性和重复性试验,建立了CSFV和PRRSV的双重RT-PCR方法。结果显示:该方法能特异检测CSFV和PRRSV的cDNA最低量分别为0.96、1.48 ng,而对猪伪狂犬病毒(PRV)和猪圆环病毒(PCV)检测结果均为阴性;利用建立的双重RT-PCR对云南省部分地区送检的174份疑似CSFV及PRRSV感染的病料进行检测显示,CSFV感染率为24.14%,PRRSV感染率为47.70%,CSFV与PRRSV的混合感染率为16.67%,检测结果与单一RT-PCR结果相一致,且具有良好的可重复性;利用免疫组织化学方法对部分检测结果加以验证,验证结果与检测结果均一致。本研究成功建立了CSFV和PRRSV双重RT-PCR检测方法,为二者的快速检测和分子流行病学调查提供了一种有效手段。In order to establish a Reverse Transcription polymerase chain reaction(RT-PCR) method for simultaneous detection of classical swine fever Virus(CSFV) and porcine reproductive and respiratory syndrome virus(PRRSV). A pair of specific primers was designed based on the conserved regions of CSFV and PRRSV. The specific amplification fragments were 356 and 546 bp, respectively. A duplex RT-PCR method for detecting CSFV and PRRSV was established by optimizing the reaction conditions and by testing its specificity, sensitivity and reproducibility. The results showed that the method specifically detected the minimum detection amounts of cDNA of CSFV and PRRSV to be 0.96 and 1.48 ng, respectively, the porcine pseudorabies virus(PRV) and porcine circovirus type 2(PCV-2) to be both negative. 174 disease materials suspected to infected with CSFV and PRRSV were collected in some areas in Yunnan Province and were tested by the established duplex RT-PCR With the results that the rate of PRRSV infection was 47.70%, the rate of CSFV infection was 24.14% and the rate of CSFV and PRRSV mixed infection was 16.67%. These results were consistent with the results of single RT-PCR detection;which indicated that our Our RT-PCR had good repeatability. Part of the detected results was verified by immunohistochemistry with results consistent with the tested results, which indicated a good reliability of our RT-PCR. The method established here provided an effective means for rapid detection, differential diagnosis and molecular epidemiological study of CSFV and PRRSV.

关 键 词：猪瘟病毒 猪繁殖与呼吸综合征病毒 双重RT-PCR 检测方法 

分 类 号：S852.6[农业科学—基础兽医学]