琼脂糖凝胶电泳与核酸印迹杂交法检测细菌毒力基因PCR产物的灵敏度比较研究
Comparison of sensitivities of agarose gel electrophoresis and nucleic acid blot hybridization in detecting bacterial virulence gene PCR product
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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6.7102 cfu(56.87 pg)/PCR体系,对SC84菌株cps2J基因PCR产物的最低检测限为 4.3102 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6.710-1 cfu(56.87 fg)/PCR体系,对SC84菌株cps2J基因PCR产物的最低检测限为4.310-1 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。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 6.7102 cfu(56.87 pg)/PCR and 4.3102 cfu(12.65 pg)/PCR. The detection limits of nucleic acid blot hybridization for stx2 and cps2J were estimated to be 6.710-1 cfu(56.87 fg)/PCR and 4.310-1 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.
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