一种新型同时检测6种猪病毒病简化靶序列富集多重PCR的建立和初步应用

Development of a novel simplified target enriched multiplex PCR for simultaneously detection of 6 porcine pathogens and its preliminary application

为建立一种新型同时检测6种猪常见病的方法,针对猪伪狂犬病病毒(PRV)、猪乙型脑炎病毒(JEV)、猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)和猪细小病毒(PPV)的保守基因,设计6对特异引物,在引物的5′端加上一段非同源性标签序列,再设计1对可识别标签序列的超级引物。通过优化反应条件,建立了6种猪常见病毒的简化靶序列富集多重PCR(Tem-PCR)检测方法。灵敏度试验结果显示,该方法对PRV、JEV、CSFV、PCV2、PRRSV、PPV核酸的最低检测限度分别为4.36×103、2.21×103、2.69×103、3.18×103、2.63×104、3.12×104copies/L,而单重PCR检测的最低限度分别为1.3×101、2.59×102、8.03×101、3.29×101、2.65×101、2.45×102copies/L。该方法对54份临床样本的检测结果与国标单重PCR检测结果的一致性为94.9%。本试验建立的方法特异、灵敏、操作简便且成本低廉,对这6种猪病常见病原的同时检测有一定的参考意义。

To establish a novel method for the detection of 6 main viral pathogens of porcine simultaneously and promote the development of Tem-PCR in the detection of animal pathogen,six pairs of primers were designed according to PRV,JEV,CSFV,PCV2,PRRSV and PPV conservative genes region,respectively.Non-homologous label sequences were introduced into 5′end of every primer,and a pair of super primers was designed to recognize the label sequences.After optimizing reaction conditions,a simplified Tem-PCR for simultaneous detection of PRV,JEV,CSFV,PCV2,PRRSV and PPV was established.sitivity test showed the minimum detection limit for the 6 viruses were up to 4.36×103,2.21×103,2.69×103,3.18×103,2.63×104,3.12×104copies/ L by a simplified Tem-PCR,and1.3×101,2.59×102,8.03×101,3.29×101,2.65×101,2.45×102copies/ L by a single PCR. 54 clinical samples detection results showed the consistency of simplified Tem-PCR and Chinese standard detection was 94.9%.This method is a rapid, specific,sensitive,simple and cheap way for single or mixed detection of the 6 viruses simultaneously.And it has great significance in the development of Tem-PCR on animal epidemic disease diagnosis in China.