细菌核酸提取方法对荧光定量PCR检测差异分析

Comparison of methods of DNA extraction for real time PCR analysis

目的观察和分析常见食源性病原菌及粪便中病原菌DNA提取方法对荧光定量PCR检测结果的影响。方法以弯曲菌、副溶血弧菌和沙门菌为模型菌株,分别采用水煮法、细胞裂解法、试剂盒法(过滤法和磁珠法)提取病原菌DNA并进行荧光定量PCR检测并对试验结果作差异比较分析;应用细胞裂解法和过滤法分别提取24份随机抽样的粪便标本DNA,针对细菌16SrDNA特异序列进行荧光定量PCR检测,比较2种DNA提取方法对于病原菌检测结果的差异性。结果在不同菌种、不同浓度的条件下,细胞裂解法提取细菌纯培养物DNA的Ct值均低于其他三种方法(P<0.05),水煮法、过滤法、磁珠法的Ct值之间的比较结果呈现多样性。细胞裂解法提取粪便标本的病原DNA为模板进行细菌16S rDNA特异序列的荧光定量PCR检测结果阳性18份,阴性6份,试剂盒过滤法提取24份粪便标本检测结果皆为阳性。结论在对纯培养的3种食源性病原菌进行荧光定量PCR鉴定时,为节省时间和成本可应用细胞裂解法或水煮法提取模板DNA,与2种商业试剂盒(过滤法和磁珠法)相比效果较好;对粪便标本病原菌进行荧光定量PCR检测时,试剂盒提取粪便标本病原DNA的扩增效果优于细胞裂解法。

Objective To compare the effects of methods of template DNA extraction for real-time PCR analysis of common food-borne pathogens from samples from pure culture bacteria or human stool. Methods Three major foodborne pathogens-Campylobacter, Vibrio parahaemolyticus, and Salmonella were used as model bacteria, the DNA templates from the pure bacteria culture were extracted via direct boiling, cell lysis, and commercial kits(filtration and immuno-magnetic beads). The DNA template from stool samples was extracted with cell lysis and a commercial method involving filtration. Real-time PCR analysis was performed with specific TaqMan probes for Campylobacter, V. parahaemolyticus, and Salmonella and 16 SrDNA from general bacteria in stool samples. Results The Ct value of DNA extracted via cell lysis was lower than that for the three other methods when using various concentrations of pure cultured Campylobacter, V. parahaemolyticus, and Salmonella. When DNA was extracted via cell lysis, real-time PCR analysis indicated that 18 stool samples tested positive and 6 tested negative. When DNA was extracted via a commercial kit, all 24 of the stool samples tested positive according to 16 SrDNA analysis. Conclusion In real-time PCR analysis, a DNA template obtained via direct boiling and cell lysis was sufficient for identification of pure culture bacteria. However, using a commercial kit for DNA extraction from stool samples might be better than other methods for real-time PCR analysis.