鸡大肠杆菌traT基因的扩增及TraT-DIG探针制备

AMPLIFICATION OF traT GENE OF E.coli IN POULTRY AND PREPARATION OF traT-DIG GENE PROBE

以致病性E .coli的质粒为模板 ,用人工合成引物扩增出致病性相关的traT基因核心部分 ;将菌株的致病性与traT基因的扩增结果进行比较 ,强致病力菌株均扩增出traT基因 ,无致病力菌株未扩增出traT基因 ,绝大多数中等致病力菌株亦扩增出traT基因。扩增获得的traT基因经纯化后标记地高辛 (Digoxin ,DIG)研制出traT DIG基因探针 ,该探针与鸡沙门氏菌、鸡白痢沙门氏菌、副伤寒沙门氏菌不发生交叉反应 ;以原位杂交检测不同血清型菌株的致病力 :强致病力菌株全部阳性 ,无致病力菌株全部阴性 ,89 7%的中等致病力菌株为阳性。对 6 0 0株分离自病死鸡实质脏器的大肠杆菌的检测 ,结果为 :84 % (50 4 / 6 0 0 )的菌株杂交阳性 ,16 % (96 / 6 0 0 )的菌株杂交阴性 ,提示从病死鸡实质脏器分离的菌株 ,少部分为无致病力菌株。

The core of traT gene,pathogenicity concerned,was amplified by using artificial synthesis primers and pure extracted plasmid of pathogenic E.coli strain as template.The results of amplification of traT gene were compared with pathogenicity of E.coli strain.traT gene was amplified among all high pathogenicity strains and most of median pathogenicity strains,no traT gene was amplified among non pathogenicity strains.traT DIG gene probe was developed after purification of amplified traT gene and labelling with Digoxin (DIG).In Situ hybridization of the probe,no cross reaction was found with S.gallinarum,S.paratyphi and S.pullorium.The pathogenicity was detected by the probe among different serotype strains.Positive results were found among all high pathogenicity and 89.7% median pathogenicity strains,and only negative result was found among non pathogenicity strains.84%(504/600) of strains showed positive and 16% of strains showed negative in detecting 600 strains isolated from internal organs of infected chickens.It suggested that small parts of non pathogenicity strains existed in strains isolated from infected chickens.