副猪嗜血杆菌aroA基因的克隆与序列分析及2种三重PCR检测方法的建立

本试验对分离的5株副猪嗜血杆菌进行了"卫星" 生长试验、生化鉴定和PCR检测。通过对aroA基因序列进行分析,发现这5株菌同标准菌株相比,其核苷酸序列相似性为96.3%~99.8%,氨基酸序列相似性为98.2%~99.5%。此外,还建立了分别针对副猪嗜血杆菌(Hps)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)及猪圆环病毒2型(PCV2)、猪细小病毒、猪伪狂犬病病毒的三重PCR检测方法;敏感性试验表明,三重PCR最低能检出Hps、PPV、PRV、PCV2的DNA分别为12、16、7.6和6.3 pg。

Isolation and Identification of Haemophilus parasuis, Cloning and Sequence Analysis of aroA Gene and Institution of Two Program of Triplex PCR Assay

In this study,5 strains of Haemophilus parasuis(Hps)were identified by ’satellite’ growing test,biochemical and PCR assay.The results showed that aroA gene of the isolated Hps had a high degree of similarity with classical culture strain in the sequence of nucleotide and amino acid sequence,which was between 96.3% to 99.8% and 98.2% to 99.5% respectively.We implemented two program of triplex PCR assay,one group was used to detect simultaneously infections of Hps,PCV2 and PPV,the other group was developed to detect the PCV2,PPV and PRV.The sensitivity tests indicated that the triplex PCR assay may be used for detecting Hps,PPV,PRV and PCV2 of 105 dilute strength;the DNA minimun detectable quantity of Hps,PPV,PRV,PCV2 was 12,16,7.6 and 6.3 pg respectively.