猪呼吸和繁殖障碍类病毒性疫病多重PCR检测方法的建立及应用

Establishment and application of multiplex PCR detection method for porcine respiratory and reproductive disorders

根据GenBank中PRRSV ORF6、PCV2 ORF2、PPV NS1、PRV gB、SIV M、JEV NS1、CSFV E2等基因的部分片段,分别设计特异性引物,用实验室已保存的病毒及阳性病料,通过对7个病毒单一PCR的退火温度和敏感性进行优化和检测,再选择合适的多重PCR缓冲体系及酶,优化退火温度、模板及引物浓度,最后检测多重PCR的敏感性、特异性及重复性,建立一种能够同时且快速鉴别PRRSV、PCV2、PPV、PRV、SIV、JEV和CSFV这7种常见猪呼吸道和繁殖障碍类病毒的多重PCR检测方法,并用其对2017-2019年贵州部分地区的150份临床病料进行检测。多重PCR特异性检测结果表明:从混合病毒基因组中分别扩增出大小为224,353,424,549,684,831,1 137 bp的特异性片段,未扩增出E.coli、APP、PEDV及正常组织的DNA/RNA;敏感性试验表明,该方法对病毒的最低检测量分别为PRRSV 94 pg、PCV2 66 pg、PPV 68 pg、PRV 20 pg、SIV 35 pg、JEV 17 pg、CSFV 14 pg;重复性试验表明:相同条件下的6次重复试验均能扩增出7个病毒的目的条带;应用试验表明:150份临床病料中二重感染阳性率高达66.00%(99/150);三重感染阳性率达11.33%(17/150);四重感染阳性率达2.00%(3/150);五重感染阳性率达0.67%(1/150);六重感染阳性率为0,七重感染阳性率为0,阳性PRRSV、PCV2、PPV、JEV、SIV的多重PCR与单一PCR的符合率均为100.00%,阳性CSFV和PRV的符合率为90.00%以上,研究建立的多重PCR检测方法具有敏感性高、特异性强、重复性好的特点,对猪场猪呼吸和繁殖障碍类病毒病混合感染的快速诊断具有十分重要的意义。

According to the partial fragments of PRRSV ORF6,PCV2 ORF2,PPV NS1,PRV gB,SIV M,JEV NS1,CSFV E2 and other genes in GenBank,specific primers were designed,and the virus and positive disease materials saved in the laboratory were used to optimize annealing temperature of single virus PCR and sensitivity was detected,and the appropriate multiplex PCR buffer system and enzyme were selected to optimize the annealing temperature,template and primer concentration,and finally the sensitivity,specificity and reproducibility of multiplex PCR were detected.To establish a multiplex PCR assay for the simultaneous and rapid identification of seven common porcine respiratory and reproductive disorders viruses such as PRRSV,PCV2,PPV,PRV,SIV,JEV and CSFV,and 150 clinical samples as a part of Guizhou from 2017 to 2019 were tested.The results of multiplex PCR-specific detection showed that specific fragments of 224,353,424,549,684,831 and 1 137 bp were amplified from the mixed virus genome,and E.coli APP,PEDV and normal tissue DNA/RNA was not amplified;sensitivity tests showed that the minimum detection of the virus was PRRSV 94 pg,PCV2 66 pg,PPV 68 pg,PRV 20 pg,SIV 35 pg,JEV 17 pg,CSFV 14 pg,respectively;reproducibility test showed that the 6 times of the same tests can amplify the target band of 7 viruses;the application test shows that the positive rate of double infection in 150 clinical materials is as high as 66.00%(99/150);the positive rate of triple infection was 11.33%(17/150);the positive rate of quadruple infection was 2.00%(3/150);the positive rate of five-fold infection was 0.67%(1/150);the positive rate of re-infection was 0,and the positive rate of seven-infection was 0.The coincidence rate of multiplex PCR and single PCR for positive PRRSV,PCV2,PPV,JEV and SIV was 100.00%,and the coincidence rate of positive CSFV and PRV was over 90.00%.The multiplex PCR assay established by the research has the characteristics of high sensitivity,specificity and good reproducibility.It is of great significance for the rapid diagnosis of mixed infection of respiratory tract and reproductive disorders in pig farms.