猪呼吸道疾病综合征4种病原菌复合PCR方法的建立及其初步应用

Development and preliminary application of quadruple PCR for four pathogenic bacteria of porcine respiratory disease complex

目的建立检测猪呼吸道疾病综合征(Porcine respiratory disease complex,PRDC)中肺炎支原体(Mycoplasmahyopneumoniae,Mhp)、猪链球菌(Streptococcus suis,SS)、副猪嗜血杆菌(Haemophilus parasuis,HPS)和猪多杀性巴氏杆菌(Pasteurella multocida,Pm)的四重PCR方法。方法根据GenBank中登录的Mhp的P36基因序列设计引物,同时参考文献分别合成SS、HPS和Pm的特异性引物,通过4因素(Taq酶、Mg2+、引物、dNTPs)4水平的L16(44)正交试验优化PCR反应体系,建立检测Mhp、SS、HPS和Pm的四重PCR方法,并进行特异性、敏感性及重复性验证。运用该方法及单一PCR方法,对PRDC猪的鼻腔拭子及肺脏组织样品进行检测,另对肺脏组织样品进行SS、HPS和Pm的常规分离鉴定。结果确立了四重PCR方法最适反应体系及反应条件;应用建立的四重PCR方法在568、294、821和457 bp处均可见与预期相符的特异性条带,8次重复扩增后的电泳结果一致,最低检测限量分别为560、260、280和200 pg,表明该方法特异性、敏感性及重复性较好;PRDC猪的鼻腔拭子及肺脏组织样品的四重PCR方法检测结果与单一PCR方法相比,差异无统计学意义(P>0.05),四重PCR及单一PCR方法的HPS的检出率则显著高于常规细菌分离鉴定(P<0.05)。结论所建立的四重PCR方法,可直接用于临床PRDC 4种病原菌的鉴定,为开展PRDC流行病学调查提供了有效的技术手段。

Objective To develop a quadruple PCR method for four pathogenic bacteria, i.e. Mycoplasma hyopneumoniae （Mhp）, Streptococcus suis （SS）, Haernophilus parasuis （HPS） and Pasteurella muhocida （Pm） of porcine respiratory disease complex （PRDC）. Methods Specific primers were designed upon the P36 gene of Mhp reported in GenBank as well as the genes of SS, HPS and Pm reported in references, based on which a quadruple PCR method was developed through optimization of reaction system by orthogona/test on four factors （Taq, Mg2＋, primers and dNTPs） at four levels, and verified for specificity, sensitivity and reproducibility. The developed method was used for determination of nasa/swab and lung tissue samples of pigs with PRDC, and for routine isolation and identification of SS, HPS and Pm in lung tissue, using single PCR as control. Results The reaction system and condition of the quadruple PCR were optimized. Specific bands at lengths of 568, 294, 821 and 457 bp respectively were amplified by the developed quadruple PCR, which were consistent with those expected. The electrophoretic results of amplified products of 8 repeat PCR were in agreement, and the minimum detection limits of Mhp, SS, HPS and Pm were 560, 260, 280 and 200 pg respectively. It indicated high specificity, sensitivity and reproducibility of the developed method. The determination results of nasal swab and lung tissue samples of pigs with PRDC by the developed quadruple PCR showed no significant difference with those by single PCR （P 〉 0. 05）. However, the positive rate of HPS determined by both quadruple and single PCR were significantly higher than that by routine isolation and identification of bacteria （P 〈 0. 05）. Conclusion The developed quadruple PCR method may be directly used for identification of four pathogenic bacteria of PRDC, which provided an effective technique for epidemiological survey of PRDC.