副猪嗜血杆菌病原检测及aroA基因PCR-RFLP分型

Detection and Genotyping of Haemophilus parasuis aroA Gene with PCR-RFLP

本试验应用PCR方法及PCR-RFLP技术对aroA基因在副猪嗜血杆菌病原检测及基因分型中的作用进行探讨。以针对aroA基因的PCR引物成功检测出18株来自广西各地区猪场的副猪嗜血杆菌,敏感性检测结果表明,该PCR方法可检测的最低菌数为102个。对该18株副猪嗜血杆菌进行aroA基因的序列测定,并进行aroA基因的酶切位点分析,筛选出HindⅢ和FokⅠ两种限制性内切酶,利用PCR-RFLP技术对1株血清5型参考菌株与本研究中18株广西菌株aroA基因的完整编码序列进行HindⅢ和FokⅠ限制酶谱分析,结果显示可分为与毒力相关的3种谱型。本研究结果表明,应用PCR方法及PCR-RFLP技术对副猪嗜血杆菌进行检测分析有助于更好地研究副猪嗜血杆菌的生物学特性及aroA基因的功能。

The PCR assay and PCR-RFLP were applied to study the function of aroA gene on the detection and genotyping of Haemophilus parasuis.18 strains of Haemophilus parasuis isolated from swine in Guangxi were detected successfully by using the PCR assay with primers targeted to aroA gene,and the sensitive test showed that the lowest amount of Hps detectable by this PCR-based test was 102.The aroA gene of the 18 strains was sequenced,and enzyme restriction analysis of the aroA sequences available was performed to define which restriction enzymes more practical.HindⅢ and FokⅠ were chosen to do RFLP analysis in all 18 Haemophilus parasuis strains and one reference strain.The result of the analysis showed that there were three patterns among the groups.The study suggests that the PCR assay and PCR-RFLP applied to detection and genotyping are useful to further research the biological characteristics and gene function of Haemophilus parasuis.