【摘要】 目的探讨核酸印迹杂交法在检测细菌毒力基因方面的应用价值。方法分别以10倍连续稀释的O157∶H7肠出血性大肠埃希菌和血清2型猪链球菌染色体提取物为模板,与志贺毒素2基因(stx2)和荚膜多糖2J基因(cps2J)特异性引物进行聚合酶链式反应(PCR)。用1%琼脂糖凝胶电泳及核酸印迹杂交法检测PCR产物,比较两种方法的最低检测限。结果 1%琼脂糖凝胶电泳对EDL933菌株stx2基因PCR产物的最低检测限为6.7×102cfu(56.87 pg)/PCR体系,对SC84菌株cps2J基因PCR产物的最低检测限为4.3×102 cfu(12.65 pg)/PCR体系;核酸印迹杂交法对EDL933菌株stx2基因PCR产物的最低检测限为6.7×10-1 cfu(56.87 fg)/PCR体系,对SC84菌株cps2J基因PCR产物的最低检测限为4.3×10-1 cfu(12.65 fg)/PCR体系。结论用核酸印迹杂交法检测肠出血性大肠埃希菌stx2基因和猪链球菌cps2J基因PCR产物的灵敏度均比普通琼脂糖凝胶电泳检测法高了1000倍。更多还原

【Abstract】 Objective To explore the significance of nucleic acid blot hybridization applying in detecting bacterial virulence gene.Methods Polymerase chain reaction(PCR) was performed by using a tenfold serial dilution chromosome template of Enterohemorrhagic E.coli O157∶H7(strain EDL933) and Streptococcus suis serotype 2(strain SC84) with the stx2-specific and cps2J-specific primer.1% agarose gel electrophoresis(AGE) and nucleic acid blot hybridization were carried out to determine the detection limits of the amplification products.Results The detection limits of 1% AGE for stx2 and cps2J were estimated to be 6.7×102 cfu(56.87 pg)/PCR and 4.3×102 cfu(12.65 pg)/PCR.The detection limits of nucleic acid blot hybridization for stx2 and cps2J were estimated to be 6.7×10-1 cfu(56.87 fg)/PCR and 4.3×10-1 cfu(12.65 fg)/PCR.Conclusion The detection limits of nucleic acid blot hybridization for stx2 and cps2J were 1000 times higher than those of routine AGE.更多还原