猪流行性腹泻病毒、传染性胃肠炎病毒、A群轮状病毒和嵴病毒多重RT-PCR检测方法的建立及临床应用  

Establishment and clinical application of a multiplex reverse transcription-PCR for rapid detection of porcine epidemic diarrhea virus,transmissible gastroenteritis virus,group A rotavirus,and porcine kobuvirus

作　　者：霍金耀[1] 郑逢梅[1] 陈陆[1] 余秋颖[1] 常洪涛[1] 陈文定[1] 明星[1] 郑关民[1] 赵军[1] 王川庆[1] 

机构地区：[1]河南农业大学,河南郑州450002

出　　处：《中国兽医学报》2013年第12期1792-1797,共6页Chinese Journal of Veterinary Science

摘　　要：本研究建立了可同时检测猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、传染性胃肠炎病毒(Transmissible gastroenteritis Virus,TGEV)、A群轮状病毒(Group A rotavirus,GARV)和猪嵴病毒(Porcine kobuvirus)的多重RT-PCR方法。检测中,建立的多重RT-PCR方法能够检测到500pg的TGEV、PEDV、GARV和猪嵴病毒等量混合RNA模板,与常规的单一RT-PCR检测结果基本相同(检测TGEV、PEDV、GARV和猪嵴病毒的灵敏性分别为100%、100%、93.33%和96.67%,特异性均为100%)。结果表明,建立的多重RT-PCR方法敏感性和特异性良好,可作为临床上猪病毒性腹泻病因快速、高效的诊断工具。应用该方法对2010-2012年华中地区190份腹泻仔猪样本进行检测,PEDV、TGEV、GARV和猪嵴病毒的阳性率分别为62.11%、0.53%、7.37%和82.11%。混合感染方面,PEDV和猪嵴病毒混合感染率为47.89%,PEDV和GARV混合感染率为4.74%,GARV和猪嵴病毒混合感染率为7.37%,PEDV、GARV和猪嵴病毒混合感染率为4.74%,未发现TGEV与其它3种病毒的混合感染情况。另外,有27份样本中仅检出PEDV(14.21%),57份样本只检出猪嵴病毒(30%)。分析表明,我国自2010年底大面积暴发的病毒性腹泻是多病原混合感染造成的,主要病原为PEDV,猪嵴病毒在其中所起作用尚待进一步验证和研究。A novel multiplex reverse transcription polymerase chain reaction （multiplex RT-PCR） was developed,which could detect porcine epidemic diarrhea virus （PEDV）, transmissible gastro- enteritis virus （TGEV）, group A rotavirus （GARV）, and porcine kobuvirus. In the laboratory test,the detection limit of multiplex RT-PCR is 500 pg mixed RNA template of TGEV,PEDV, GARV and porcine kobuvirus. The relative sensitivity and specificity of multiplex RT-PCR were e- qual to conventional single RT-PCR assays （sensitivity： 100%, 100%,93. 33%, and 96. 67% for TGEV,PEDV,GARV,porcine kobuvirus,repectively specificity. 100% for all 4 viruses）. The re- sults indicate that the multiplex RT-PCR with high sensitivity and specificity provides a rapid and cost-effective etiological diagnostic tool for viral gastroenteritis in piglets. A total of 190 fecal and intestinal samples from piglets with acute gastroenteritis were collected in central china during Oc- tober 2010 and August 2012 ,which were tested by the established multiplex RT-PCR. The positive rates of PEDV,TGEV,GARV,and porcine kobuvirus are 62.11% ,0.53% ,7.37% ,and 82.11%,respectively. The coinfection rates of PEDV/porcine kobuvirus, PEDV/GARV, GARV/porcine kobuvirus,PEDV/GARV/porcine kobuvirus are 47.89 %, 4.74 %, 7.37% and 4.74 ~, respective- ly, however there is no coinfection of TGEV with other virus. In addition, single infection of PEDV and porcine kobuvirus was detected in 27 samples （14. 25%） and 50 samples （30%） with this method, respectively. The results show that viral gastroenteritis of piglets in central china since the end of 2010 is closely related to coinfection of diarrhea-related virus,of which PEDV is the leading cause. The role of porcine kobuvirus needs further confirmation and research.

关 键 词：多重RT—PCR PEDV TGEV GARV 猪嵴病毒 

分 类 号：S852.65[农业科学—基础兽医学]