多重聚合酶链式反应-毛细管电泳技术检测5种食源性致病菌

Detection of 5 kinds of foodborne pathogens by multiplex polymerase chain reaction and capillary electrophoresis technology

中文关键词:  多重聚合酶链式反应  毛细管电泳  食源性致病菌  检测方法

英文关键词:multiplex polymerase chain reaction  capillary electrophoresis  foodborne pathogens  detection method

中文摘要:

      目的 利用多重聚合酶链式反应(polymerase chain reaction, PCR)-毛细管电泳技术, 构建一种快速检测沙门氏菌、大肠埃希氏菌O157:H7、单核细胞增生李斯特氏菌、金黄色葡萄球菌和副溶血性弧菌的方法。方法 针对5种食源性致病菌, 选择保守性较好的致病基因设计特异性多重PCR引物, 利用毛细管电泳成像分析多重PCR产物。通过对PCR及电泳条件进行优化, 建立5种食源性致病菌的多重PCR-毛细管电泳检测方法, 并对其特异性、灵敏度进行研究。结果 5对引物特异性良好, 均未出现非特异性扩增, 多重PCR最佳引物浓度为0.2 μmol/L, 最佳退火温度为56.0 ℃, 毛细管电泳最佳分离模式为AM900, 灵敏度较高, 检测灵敏度可达到5×10–3 ng/μL。结论 本研究建立的多重PCR-毛细管电泳方法用于检测5种食源性致病菌, 操作简便、特异性和灵敏度高, 在食源性致病菌检验中具有较好的实用价值。

英文摘要:

      Objective To develop a rapid method for detecting Salmonella, Escherichia coli O157, Listeria monocytogenes, Staphylococcus aureus, and Vibrio parahaemolyticus using multiplex polymerase chain reaction (PCR) and capillary electrophoresis technology. Methods Specific multiplex PCR primers were designed using conserved pathogenic genes of 5 kinds of foodborne pathogens, and the products were analyzed using capillary electrophoresis imaging. By optimizing the PCR reaction and electrophoresis conditions, a multiplex PCR capillary electrophoresis detection method was established. The specificity and sensitivity were studied as well. Results The specificity of the 5 pairs of primers was acceptable and no non-specific amplification occurred. The optimal primer concentration for multiplex PCR reaction was 0.2 μmol/L, and the optimal annealing temperature was 56.0 ℃. The optimal separation mode for capillary electrophoresis was AM900. The method had a high sensitivity, and the detection sensitivity could reach 5×10–3 ng/μL. Conclusion The multiplex PCR capillary electrophoresis method for detecting 5 kinds of foodborne pathogens established in this study is simple for operation with high specificity and sensitivity, and can be very practical in detection of foodborne pathogens.