多重PCR鉴定猪链球菌2型毒力菌株

Detection of Virulent Streptococcus suis Serotype 2 by Multiplex PCR

为了鉴定猪链球菌2型毒力菌株,根据猪链球菌2型(Streptococcus suis serotype 2,SS2)的荚膜多糖基因(capsular polysaccharide 2J,cps2J)、溶菌酶释放蛋白基因(muramidase-released protein,mrp)、毒力相关序列orf2基因(virulent-associate sequence orf2)设计合成3对引物,建立三重PCR检测方法,并对PCR扩增产物进行测序、酶切、特异性及敏感性实验。结果显示:26株SS2致病菌株均同时扩增出这3个目的片段,5株其他血清型猪链球菌(SS1,SS7,SS9)及13株非猪链球菌的其他猪源链球菌(兰氏B群、C群和D群)均未扩增出cps2J和mrp片段,检测结果与菌株的已知毒力因子背景完全相符。26株SS2的扩增片段经HincⅡ酶切鉴定,与预期结果一致。以SS2四川株ZY05719为模板的三重PCR产物(cps2J,mrp,orf2)测序结果与GenBank上相关序列比较,同源性分别为99%、100%、94%。该PCR敏感极限是600个细菌/每个反应。本试验所建立的三重PCR,特异性强,敏感性好,可用于实验室的快速诊断。

A multiplex PCR was developed for rapid detection of virulent Streptococcus suis semtype 2 strains. Three distinct DNA targets were amplified. The primers were designed based on the serotype 2 （and 1/2） specific cps2J gene, the muramidase-released protein（mrp） and virulence-associated sequence off2. The assays including Sequence analysis and restriction endonuclease analysis and sensitivity and specificity test, were also done. Three targets can be amplified in all virulent SS2 isolates ,while, both targets of mrp and cps2J can not be amplified in 5 other serotypes isolates（SS1, SS7, SS9）and 13 other Streptococcal isolates （group B, C and D）. The results of restriction endonuclease analysis were in accordance with the expected. The Blast results showed that the PCR products （cps2J, mrp, orf2）of Sichuan isolate ZY05719 has 99%, 100% and 94% homology to that of the correlated sequences in GenBank. The multiplex PCR n,ethod was highly sensitive and specific. It can be used for rapid diagnosis and survey of SS2.