猪呼吸道病原菌多重PCR检测方法的建立及应用

Establishment and Application of Multiplex PCR for Detection of Pathogenic Bacteria in Porcine Respiratory Tract

为建立一种快速诊断由猪链球菌(SS)、猪胸膜肺炎放线杆菌(App)、猪多杀性巴氏杆菌(Pm)、副猪嗜血杆菌(Hps)、猪支气管败血波氏杆菌(Bb)和猪肺炎支原体(Mhp)单独或混合感染引起的猪呼吸道疾病的检测方法,基于上述6种病原菌对应的gdh(SS)、apxⅣA(App)、KMT1(Pm)、16S rRNA(Hps)、fla(Bb)和p36 ldh(Mhp)基因设计特异性引物,通过优化各反应条件建立了能同时检测SS、App、Pm、Hps、Bb和Mhp的多重PCR方法。结果显示,该方法特异性强,仅对SS、App、Pm、Hps、Bb和Mhp有特异性扩增,对大肠埃希氏菌、奇异变形杆菌、停乳链球菌和金黄色葡萄球菌等其他病原菌的扩增结果均为阴性;敏感性较高,对6种病原菌DNA的最低检出限分别为SS 8.66×10^(2)拷贝/μL、App 1.05×10^(4)拷贝/μL、Pm 8.61×10^(2)拷贝/μL、Hps 9.01×10^(2)拷贝/μL、Bb 7.54×10^(2)拷贝/μL、Mhp 3.86×10^(3)拷贝/μL。利用该多重PCR和各单项PCR分别检测贵州省9个市(州)部分猪场的469份临床样品,结果显示,多重PCR阳性检出率为App 0.85%(4/469)、Pm 15.35%(72/469)、SS 59.49%(279/469)、Hps 52.67%(247/469)、Mhp 6.40%(30/469)、Bb 5.12%(24/469)。混合感染主要以SS和Hps为主,阳性检出率为30.92%(145/469),其次是Pm、SS和Hps混合感染,阳性检出率为7.04%(33/469),其他混合感染情况占比较低。多重PCR和单项PCR的总阳性符合率为96.76%。结果表明,建立的多重PCR检测方法为SS、App、Pm、Hps、Bb和Mhp混合感染的临床快速检测及流行病学调查提供了技术支持。

To establish a rapid and accurate detection method for the diagnosis of respiratory diseases in pigs caused by single or mixed infection of Streptococcus suis(SS),Actinobacillus pleuropneumoniae(App),Pasteurella multocida(Pm),Hemophilus parasuis(Hps),Bordetella bronchisepticus(Bb)and Mycoplasma hyopneumoniae(Mhp),amultiplex PCR method was established that can simultaneously detect SS,App,Pm,Hps,Bb and Mhp by designing the specific primers based on gdh(SS),apxⅣA(App),KMT1(Pm),16S rRNA(Hps),fla(Bb)and p36 ldh(Mhp)genes corresponding to the above six pathogens and optimizing reaction conditions in this study.The results showed that this method was highly specific,only for SS,App,Pm,Hps,Bb and Mhp,and negative for E.coli,Proteus mirabilis,Streptococcus lactis and Staphylococcus aureus.The sensitivity is high,and the lowest simultaneous detection limits of six pathogenic bacteria DNA templates are SS 8.66×10^(2)copies/μL,App 1.05×10^(4)copies/μL,Pm 8.61×10^(2)copies/μL,Hps 9.01×10^(2)copies/μL,Bb 7.54×10^(2)copies/μL,Mhp 3.86×10^(3)copies/μL,respectively.469 clinical samples from some pig farms of 9 cities(prefectures)in Guizhou province were detected by this multiplex PCR and each single PCR method.The results showed that the positive detection rates of multiplex PCR were 0.85%(4/469)for App,15.35%(72/469)for Pm,59.49%(279/469)for SS,52.67%(247/469)for Hps,6.4%(30/469)for Mhp,and 5.12%(24/469)for Bb.The mixed infection was mainly caused by SS and Hps,with a positive detection rate of 30.92%(145/469),followed by Pm,SS and Hps,with a positive detection rate of 7.04%(33/469),and the proportion of other mixed infections was lower.The total positive coincidence rate of multiplex PCR and single PCR was 96.76%.The multiplex PCR detection method established in this study provides technical support for rapid clinical detection and epidemiological investigation of mixed infections of SS,App,Pm,Hps,Bb and Mhp.