多重 PCR 检测副猪嗜血杆菌毒力相关三聚自转运蛋白

摘要

In order to develop a rapid multiple PCR method to detect virulence-related trimeric autotrans-porters ( VtaA) from Haemophilus parasuis ( HPS) , 2 pairs of specific primers were designed and synthe-sized using HPS VtaA VtaA1 and VtaA3 genes as templates, the multiple PCR amplification and reaction conditions were optimized, and the sensitivity and specificity of the multiple PCR were analyzed.The re-sults showed that the developed multiple PCR method with 3.4 ×107 cfu/mL of the minimum detection limit to HPS could amplify 406 and 291 bp fragments corresponding to the HPS VtaA1 and VtaA3 loci, re-spectively, but could only detect 300 bp fragment on HPS VtaA1 and 400 bp on HPS VtaA3, it did not work on detection of Actinobacillus pleuropneumoniae, Salmonella suipestifer, Escherichia coli, Brodetella bronchiseptica and Streptococcus suis.It could be concluded that this multiple PCR method had a strong specificity and a high sensitivity and could be used in rapid diagnosis and detection of HPS.%为建立快速检测副猪嗜血杆菌 ( Haemophilus parasuis, HPS) 毒力相关三聚自转运蛋白 ( Trimeric autotrans-porters, VtaA) 的多重PCR方法, 根据HPS VtaA VtaA1和VtaA3基因为模板, 设计合成了2对特异性引物, 进行多重PCR扩增及反应条件的优化, 并对多重PCR扩增的敏感性和特异性进行了分析.结果表明: 所建立的多重PCR检测方法对 HPS VtaA1 和VtaA3 基因分别能扩增出406 和291 bp 的目的条带, 最小检测限为3.4 ×107 cfu/mL; 此多重PCR方法只能检出HPS VtaA1和VtaA3基因400和300 bp大小的目的条带, 而不能检出胸膜肺炎放线杆菌 ( Actinobacillus pleuropneumoniae)、 猪霍乱沙门氏菌 ( Salmonella suipestifer)、 大肠埃希氏杆菌 ( Esche-richia coli)、 支气管败血波氏杆菌(Brodetella bronchiseptica) 和猪链球菌 (Streptococcus suis).由此证明, 所建立的多重PCR方法操作简单、 快速、 特异性强和敏感性高, 能够实现对HPS的快速诊断与检测.