【作者】 白昀；

【导师】 刘棋； 罗廷荣；

【作者基本信息】 广西大学 ， 预防兽医学， 2008， 硕士

【摘要】 本研究建立了猪链球菌2型四个主要毒力因子Cps、MRP、EF、Sly的四重PCR检测方法,实验证明其特异性好,可检测的最低菌量为20CUF/ml,适合样品的大规模检测。应用以上建立的毒力因子四重PCR检测方法对138份广西猪链球菌2型菌株进行了检测,结果表明,127株分离自健康猪扁桃体的菌株的主要毒力基因型分属为:mrp+epf+sly+和mrp+epf+sly-共104株,占81.9%,mrp+epf-sly+有1株,mrp+epf-sly-有12株,mrp-epf-sly-有10株,共占18.1%。11株分离自病死猪器官的猪链球菌2型菌株毒力基因型都为mrp+epf+sly+。同时,对56株MRP基因PCR扩增呈阳性的菌株进行了部分测序,通过对核苷酸和推导氨基酸的同源性以及遗传进化系统树的分析,将这56个菌株分为2个基因型,即:Ⅰ型和Ⅱ型。Ⅰ型中包括:GX01、GX20-2、GX4Lin、GX6Lin、GXLP5、GXB30、GXB45、GXB102、GXB141、GXBM20、GXCDX23、GXCDX49、GXDL9、GXDL36、GXFS26、GXG42、GXG121、GXGG34、GXGL462、GXLB72、GXLB131、GXLP26、GXLS53、GXQL31、GXQP46、GXR147、GXRA63、GXWX193、GXWX209、GXWZ80、GXY134、GXY153、GXY160和GXZ22共34株。Ⅱ型包括:GX2Lin、GX11Lin、GXB74、GXB70、GXCLZ192、GXG6、GXGG35、GXGL547、GXHP99、GXJZ26、GXLJ122、GXQL11、GXQL25、GXR128、GXRA32、GXW32、GXW45、GXWX400、GXWZ241、GXYL12和GXYL16、GX12Lin共22株。Ⅱ型又可分成Ⅱa亚型和Ⅱb亚型,Ⅱa亚型包括:GX2Lin、GX11Lin、GXB74、GXB70、GXCLZ192、GXG6、GXGG35、GXGL547、GXHP99、GXJZ26、GXLJ122、GXQL11、GXQL25、GXR128、GXRA32、GXW32、GXW45、GXWX400、GXWZ241、GXYL12和GXYL16共21株。GX12Lin归为Ⅱb亚型。Ⅰ型中34株菌株有29株来自健康猪、5株来自发病猪,它们的核苷酸同源性在98.8%-99.9%之间,与江苏人源毒株98015核苷酸的同源性在99.0%-100%之间。22株Ⅱa亚型菌株中有20株来自健康猪、2株来自发病猪,它们的核苷酸同源性在98.0%-99.7%之间,与江苏人源毒株98015、71.9%-73.0%之间,Ⅱb亚型的GX12Lin株与Ⅱa亚型菌株的核苷酸同源性在82.5%-83.4%之间,江苏和四川人源毒株的核苷酸同源性在71.7%-72.5%之间,Ⅰ型菌株与Ⅱ型菌株核苷酸的同源性在71.1%-72.9%之间。更多还原

【Abstract】 A sensitive and specific mμltiplex PCR assay for virulence-associated factors which including CPS、MRP、EF and SLY gene of Streptococcus suis 2 was established in this study.The target strain of serotype of Streptococcus suis 2 coμld be detected definitely when the template contained as few as 20 cfu. Use this mμltiplex PCR assay,138 Streptococcus suis 2 isolates from Guangxi were detected.The resμlts confirmed that in 127 isolates from slaughtered healthy pig’s tonsil,there are 104(81.9%)strains belong to mrp+epf+sly+ and mrp+epf+sly-,1 strain belongs to mrp+epf-sly+,12 strains belong to mrp+epf-sly-and 10 strains belong to mrp-epf-sly-.At the same time,56 strains which have MRP gene were cloned and partial sequenced.The resμlts showed all this strains coμld be divided into two genotypes asⅠandⅡ,genotypeⅡcoμld be divided into 2 sub-genotypes asⅡa andⅡb.34 strains GX01,GX20-2, GX4Lin,GX6Lin,GXLP5,GXB30,GXB45,GXB102,GXB141,GXBM20, GXCDX23,GXCDX49,GXDL9,GXDL36,GXFS26,GXG42,GXG121, GXGG34,GXGL462,GXLB72,GXLB131,GXLP26,GXLS53,GXQL31, GXQP46,GXR147,GXRA63,GXWX193,GXWX209,GXWZ80,GXY134, GXY153,GXY160 and GXZ22 belong to genotypeⅠ;22 strains GX2Lin, GX11Lin,GXB74,GXB70,GXCLZ192,GXG6,GXGG35,GXGL547, GXHP99,GXJZ26,GXLJ122,GXQL11,GXQL25,GXR128,GXRA32, GXW32,GXW45,GXWX400,GXWZ241,GXYL12,GXYL16 and GX12Lin belong to genotypeⅡ,of which GX2Lin,GX11Lin,GXB74,GXB70, GXCLZ192,GXG6,GXGG35,GXGL547,GXHP99,GXJZ26,GXLJ122, GXQL11,GXQL25,GXR128,GXRA32,GXW32,GXW45,GXWX400, GXWZ241,GXYL12 and GXYL16 are in sub-genotypesⅡa,GX12Lin is in sub-genotypesⅡb.There were 29 strains isolated from healthy pigs and 5 strains isolated from diseased pigs in genotypeⅠ,the nucleotide homologies of them were 98.8%to 99.9%.In sub-genotypeⅡa,there were 20 strains isolated from healthy pigs and 2 strains isolated from diseased pigs,the nucleotide homologies of them were 98.0%to 99.7%.The nucleotide homologies between genotypeⅠandⅡwas 71.1%to 72.9%.更多还原