猪链球菌2型和9型菌株的多重PCR检测

目的建立一种快速、特异且敏感的检测方法。方法以猪链球菌2型和9型的荚膜多糖抗原编码基因簇中的cps2J和cps9H基因、猪链球菌2型的主要毒力因子编码基因epf和mrp为靶基因设计了四对引物，建立了检测猪链球菌的多重PCR反应体系。用编码cps2J和cps9H基因的引物可对猪链球菌2型和9型进行分型，用编码epf和mrp基因的引物可鉴定猪链球菌2型的主要毒力相关基因。用该多重PCR反应体系对10株猪链球菌分离株进行了鉴定，并以其它几株阴性对照菌株进行对照，检测了该反应体系的特异性。以新鲜培养的猪链球菌2型菌株进行倍比稀释后进行菌落计数，并将之作为模板．对该多重PCR反应体系检测的敏感性进行了鉴定。结果10株猪链球菌分离株中有5株是9型菌株，另有5株是2型菌株，2型菌株有2种基因型：3株cps2^ mrp^ 型，2株cps2^ mrp^-epf^-型。同时还表明该多重PCR反应体系有高度的特异性和敏感性，当模板含量在10cfu时仍能检出目的菌株。结论该多重PCR反体系是一种检测和鉴定猪链球菌2型的快速、特异且敏感的方法。

Multiplex PCR assay for the detection of serotype 2 and 9 of Streptococcus suis

To develop a highly specific, sensitive and rapid method for the detection of Streptococcus suis with multiplex PCR, 4 pairs of primers were designed, in which 2 pairs of them were based on capsular polysaccharide genes specific for serotype 2 and 9 (cps2 and cps9), and the other 2 pairs of primers were based on the virulence genes epf and mrp, encoding the extracellular factor protein (EF) and muramidase released protein (MRP) of the virulent serotype 2 respectively. Ten strains of Streptococcus suis in which 5 strains belonged to serotype 9 and the other 5 strains belonged to serotype 2 were identified by means of the multiplex PCR assay with several strains of negative controls. It was found that of the 10 strains of S.suis, isolated from diseased pigs, all were positive by the multiplex PCR assay. The 5 strains of serotype 2 could be further divided into 2 genotypes, i.e.cps2~+mrp~+epf~+ (3 strains); cps~+mrp~-epf~-(2 strains). By using the same technique of multiplex PCR, no PCR product could be detected from S.suis group C and group D, ETEC O148, STEC O138, Staphylococcus and Salmonella enteritidis.The target strain of serotype of S.suis could be detected definitely when the template contained as few as 10 cfu. It is concluded that this multiplex PCR assay is a highly specific, sensitive and rapid method for the detection of serotype of S.suis.