非洲猪瘟病毒、猪圆环病毒2型和猪圆环病毒3型三重PCR检测方法的建立

Establishment of a Triple PCR for Simultaneous Detection of ASFV,PCV2 and PCV3

为建立可同时鉴别检测非洲猪瘟病毒(ASFV)以及猪圆环病毒2型(PCV2)、3型(PCV3)的三重PCR方法,根据GenBank中登录的ASFV、PCV2、PCV3相关基因保守序列,分别设计合成特异性引物,通过对引物浓度、退火温度、循环数等反应条件进行优化,建立了针对3种病原的三重PCR检测方法,并开展了敏感性、特异性及重复性试验。结果显示:引物最佳终浓度为0.375pmol/μL,最佳退火温度为61℃,最佳循环数为35;该方法对ASFV、PCV2、PCV3的扩增目的条带分别为873、530和217bp,对猪细小病毒和伪狂犬病病毒的扩增结果均为阴性;对ASFV、PCV2、PCV3重组质粒标准品的检出下限均为1×10^(4)copies/μL;相同条件下重复性试验获得均匀一致的结果。结果表明,本研究建立的三重PCR检测方法具有特异性强,敏感性及重复性好等优点,可用于ASFV、PCV2和PCV3感染的快速鉴别诊断及流行病学调查。

In order to establish a triple PCR assay for differential diagnosis of African swine fever virus(ASFV),porcine circovirus type 2(PCV2)and type 3(PCV3),specific primers were respectively designed and synthesized for amplification of ASFV,PCV2 and PCV3 related genes based on their conserved sequences registered in GenBank,the reaction conditions such as primer concentration,annealing temperature,and number of cycles were optimized to establish a triple PCR assay for the three pathogens,followed by sensitivity,specificity and repetitive tests.The results revealed that the optimal final concentration of primers was 0.375 pmol/μL,the optimal annealing temperature was 61 ℃ and the optimal number of cycles was 35;its amplification target bands for ASFV,PCV2 and PCV3 were 873,530 and 217 bp,respectively,and negative for porcine parvovirus and pseudorabies virus;the detection limits were all 1×10^(4)copies/μL for ASFV,PCV2 and PCV3 recombinant plasmid standard substances;uniform and consistent results were obtained by the repeatability test under the same conditions.In conclusion,the established method had the advantages of strong specificity,sensitivity and repeatability,and could be used for rapid differential diagnosis and epidemiological investigation of ASFV,PCV2 and PCV3 infection.