【作者】 张坤；

【导师】 何启盖；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 在寒冷季节,猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪A群轮状病毒(PGAR)是引起猪腹泻的主要病原,其临床症状、病理变化和流行病学极为相似,在临床和组织病理学上很难区分。本试验建立了能同时检测猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪A群轮状病毒(PGAR)的多重RT-PCR检测方法。根据GenBank登录的PEDV M基因、TGEV N基因、PGAR VP7基因,利用软件Primer 5.0设计合成三对特异性引物。经条件优化,我们建立了一种能够同时检测PEDV, TGEV和PGAR的多重RT-PCR的方法,并将这种方法和常规的RT-PCR进行了比较。特异性试验中,多重RT-PCR检测方法能够检测到35 pg的TGEV-PEDV-PGAR三联苗的混合RNA。在临床检测中,应用该方法检测了华中地区75份腹泻猪粪样,同时利用常规RT-PCR检测方法对该检测方法的敏感性和特异性进行了分析(PEDV、TGEV、PGAR敏感性分别为92%、100%、100%,特异性均为100%)。该方法敏感性高、特异性强,可用于腹泻样品中PEDV、TGEV和PGAR的检测。利用已建立的PEDV、TGEV、PGAR多重RT-PCR检测方法对2009年1月至2010年2月来自湖北、河南、湖南、江西、广西等地区部分养殖场的197份猪腹泻粪样进行了检测。结果为PEDV感染58份,占29.44%；TGEV感染1份,占0.51%；PGAR感染15份,占7.61%。哺乳仔猪、保育及育肥猪和母猪三个生长阶段进行了统计分析：在PEDV 58份阳性样品中哺乳仔猪占24.14%、保育及育肥猪占55.17%、母猪占20.69%; TGEV仅从哺乳仔猪检出、保育及育肥猪和母猪未检测出；PGAR 15份阳性样品中哺乳仔猪占13.33%、保育及育肥猪占6.67%、母猪占80.00%。检测结果显示：在寒冷季节,在本次收集的腹泻样品中猪流行性腹泻病毒(PEDV)是引起不同生长阶段猪病毒性腹泻最常见的病原,其次是猪A群轮状病毒。将三份轮状病毒阳性的腹泻猪粪样接种MA104细胞进行了轮状病毒分离,盲传至第三代开始出现明显细胞病变(CPE),将分离毒株依次命名为：TM-a毒株、WH-a毒株和GX-a毒株。对分离病毒进行了常规RT-PCR鉴定；同时依据GenBank发表的猪A群轮状病毒的VP6和VP7基因序列各设计了一对引物,对三株分离毒株的VP6和VP7基因序列进行了克隆及序列分析。结果显示TM-a株VP6基因与中国武汉人源A群R479毒株亲缘关系更为接近(94.4%),而与其VP7基因序列亲缘性最近的是印度人源A群RMC321毒株(95.1%)；WH-a株VP6基因与中国北京人源A群LL3354毒株亲缘关系更为接近(96.0%),而与其VP7基因亲缘性最近的的是印度人源A群RMC321毒株(94.6%)；GX-a株VP6基因与泰国猪源A群CMP74/01毒株的亲缘性最近(94.4%)。这些进一步证实分离的三株病毒为A群猪轮状病毒以及轮状病毒的多样性和复杂性。更多还原

【Abstract】 In winter, porcine epidemic diarrhea virus(PEDV),porcine transmissble gastroenteritis virus(TGEV) and porcine group A rotavirus(PGAR) are major agents in viral diarrhea of pigs.the three viruses induce similar clinical signs,lesions and epidemiology. To develop a multiplex reverse transcription polymerase chain reaction (mutiplex RT-PCR) for detection of porcine epidemic diarrhea virus (PEDV), porcine transmissble gastroenteritis virus(TGEV) and porcine group A rotavirus(PGAR). Three pairs of primers targeting the M gene, N gene, VP7 gene of PEDV, TGEV, PGAR were designed respectively. Using the three pairs of primes, A multiplex reverse transcription polymerase chain reaction (mutiplex RT-PCR) was developed.And we compared the mutiplex RT-PCR with routine RT-PCR in the field trail.In the laboratory test, we found that the detection limit of multiplex RT-PCR is 35 pg RNA of TGEV-PEDV-PGAR vaccine. And in the field trail, a total of 75 fecal specimens from pigs with diarrhea were collected in the central area of China. The relative sensitivity and specificity of multiplex RT-PCR were evaluated. The results suggesting that this assay is equal in quality to routine RT-PCR assys (sensitivity: 92%,100%,100% for PEDV, TGEV, PGAR respectively; specificity:100% for all 3 viruses). The results indicated that the multiplex RT-PCR with high sensitivity and specificity provided a new and alternative tool for the detection of PEDV, TGEV and PGAR.A tatal of 197 fecal samples from swine with diarrheal were collected from HuBei, HeNan, HuNan, JiangXi and GuangXi and tested by a mutiplex RT-PCR that can detect PEDV, TGEV and PGAR. Fifty-eight (29.44%) infections were caused by PEDV, one (0.51%) infection was caused by TGEV and fifteen (7.61%) infections were caused by PGAR. In the PEDV and PGAR positive samples, the suckling piglets,the weaning or growing pigs and the sow accounts for 24.14%,55.17%,20.69% and 13.33%,6.67%, 80.00%. TGEV was only detected in the suckling piglets. The results indicated that porcine epidemic diarrhea virus (PEDV) and porcine group A rotavirus(PGAR) are major agents in enteric diseases of pigs.Three strains of porcine group A rotavirus(PGAR) named as TM-a, WH-a and GX-a were isolated from fecal specimens of pigs with diarrhea by using MA 104 cell, and produced obvious cytopathic effects. And these viruses were identified by routine RT-PCR.Tow pairs of primers were designed according to VP6 and VP7 sequences of porcine group A rotavirus in GenBank, and VP6 and VP7 gene were amplified by RT-PCR and cloned into pMD18-T vectors. Gene sequences analysis showed that VP6 and VP7 gene sequences of TM-a were more closely related to human rotavirus R479 isolated from WuHan and human rotavirus RMC321 isolated from India; VP6 and VP7 gene sequences of WH-a were more closely related to human rotavirus LL3354 isolated from BeiJing and human rotavirus RMC321 isolated from India; VP6 gene sequence of GX-a was more closely related to porcine rotavirus CMP74/01 isolated from Thailand. These indicated that rotavirus was diversity and complexity.更多还原