摘要
由猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)和猪繁殖与呼吸综合征病毒(PRRSV)引起的繁殖障碍性疫病在中国的不断暴发,增加了猪的发病率和死亡率。为了建立同时检测这3种病毒的多重PCR方法,本研究根据GenBank中这3种病毒的参考基因序列设计引物,并对反应中的影响因素进行优化,建立了同时检测CSFV、PRV和PRRSV的多重PCR方法。该方法扩增的基因片段大小分别为570(CSFV),232 (PRRSV)和173bp(PRV)。敏感性和特异性试验结果显示,该方法对3种病毒的核酸最低检出量分别为23.88(CSFV),13.10(PRRSV)和14.60pg(PRV),对大肠杆菌(Ec.oli)、沙门菌(Salmonella)、猪乙型脑炎病毒(JEV)及猪圆环病毒2型(PCV2)的检测结果均为阴性。对2015年5月至2016年1月收集的168份临床样本检测结果显示,CSFV阳性率为24.4%,PRRSV阳性率为21.4%,提示河北省该段时间内引起猪繁殖障碍性疫病的主要病原为CSFV和PRRSV。经多重PCR检测为PRRSV、PRV或CSFV阳性的临床样本,再次使用商品化的试剂盒进行检测,符合率为100.0%。这些结果表明,本试验建立的多重PCR方法省时、高效,可用于PRRSV、PRV和CSFV感染的临床诊断。
Outbreaks of reproductive failure disease in swine caused by classical swine fever virus(CSFV),porcine pseudorabies virus(PRV)and porcine reproductive and respiratory syndrome virus(PRRSV)have increased the morbidity and mortality of pig in China.We developed a novel multiplex assay based on reverse transcription and polymerase chain reaction(PCR)for the simultaneous detection of CSFV,PRV and PRRSV.The multiplex PCR assay produced 570,232 and173 bp amplicons for CSFV,PRRSV and PRV,respectively.The sensitivity of the multiplex PCR assay was evaluated using extracted nucleic acids containing the target sequences,and the detection limits were 23.88,13.10,and 14.60 pg for CSFV,PRRSV and PRV,respectively.A total of 168 clinical samples which were collected from aborted and dead fetuses during May 2015 to January2016 were detected using the multiplex PCR assay.The CSFV-positive rate was 24.4% and PRRSV-positive rates was 21.4%,suggesting that CSFV and PRRSV were the major causes of swine reproductive disease in Hebei province during that period.The positive samples of PRRSV,PRV or CSFV were re-detected by commercial Kits.The detect results were the same as that of the multiplex PCR.The developed multiplex PCR assay is an efficient and time saving method,and can be used for the clinical diagnosis of PRRSV,PRV and CSFV infection.
引文
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