聚合酶链式反应-变性高效液相色谱法检测5种食源性致病菌

目的建立聚合酶链式反应与变性高效液相色谱(polymerase chain reaction-denaturing high-performanceliquid chromatography,PCR-DHPLC)相结合的方法,快速检测5种食源性致病菌(沙门菌、副溶血性弧菌、福氏志贺菌、大肠埃希菌O157∶H7和单核细胞增生李斯特菌)。方法针对16S rRNA基因保守区设计引物,PCR扩增产物用变性高效液相色谱仪检测,并进行敏感性、特异性、检出率等指标测定。结果柱温61.4℃时,5种致病菌PCR产物分别呈现特异DHPLC色谱图,保留时间均为7min左右。对沙门菌、副溶血性弧菌和单核细胞增生李斯特菌检出限均为5～10CFU/ml,福氏志贺菌和大肠埃希菌O157∶H7均为1～5CFU/ml。对83株目的分离株的检出符合率为100%,38株非目的分离株检测均为阴性;对人工污染食品中的5种致病菌均可正确检出。结论该PCR-DHPLC方法具有较高的敏感性和特异性,可用于食品中5种食源性致病菌的高通量快速检测。

Detection of Five Foodborne Bacterial Pathogens by Using PCR-DHPLC

Objective To establish a PCR- DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) method for the detection of five foodborne pathogenic bacteria (Salmonella spp., Vibrio parahaemolyticus, Escherichia coli O157:H7, and Listeria monocytogenes). Method The primer sets for the conserved region of 16S rRNA gene were designed and used for PCR amplification. PCR products were detected by DHPLC, and the sensitivity and specificity of this method were tested as well. Results The five PCR products were shown as specific peak profiles with the retention time of 7 min at an oven temperature of 61.4°C. The detection limits of Salmonella spp., Vibrio parahaemolyticus, and Listeria monocytogenes were 5-10 CFU/ml, while that of Shigella flexneri and Escherichia coli O157:H7 were 1-5 CFU/ml. All 83 target bacteria isolates tested were correctly identified and all 38 non-target strains tested were negative. The five pathogens in artificially contaminated food samples were also correctly identified by this method. Conclusion The PCR-DHPLC method was specific and sensitive for the detection of these five bacterial pathogens and could be used for quickly detecting a large number of samples.