干燥型荧光PCR试剂盒检测食源性致病菌研究

Study on the detection of food-borne pathogens by freeze-dried real-time PCR kits

目的研究干燥型荧光PCR试剂盒检测副溶血性弧菌、大肠埃希氏菌O157:H7和单增李斯特菌3种食源性致病菌的灵敏度、特异性和检测人工污染样品情况。方法目标菌和非目标菌接种至血平板, 36℃培养过夜,目标菌比浊至0.5 McF(麦氏单位), 10倍连续稀释后提取DNA,进行灵敏度研究;非目标菌直接提取DNA后检测,进行特异性研究;用高、中、低3个浓度目标菌液人工污染食品样品,按国标方法培养后用干燥型荧光PCR试剂盒检测。结果副溶血性弧菌、大肠埃希氏菌O157:H7和单增李斯特菌的最低检测浓度分别为140、380和7900 CFU/mL。3种目标菌人工污染食品样品的最低检测浓度分别为1.2 CFU/25 g、5.1CFU/25 g、10 CFU/25 g。每种试剂盒仅对目标菌株的检测结果呈阳性,非目标菌株均呈阴性。结论新型干燥型荧光PCR检测试剂盒具有较好的灵敏度和良好的特异性,适用于食品中副溶血性弧菌、大肠埃希氏菌O157:H7和单增李斯特菌的检测。

Objective To study the sensitivity and specificity of determination of Vibrio parahaemolyticus,Escherichia coli O157:H7 and Listeria monocytogenes by the freeze-dried real-time PCR kits and determine 3 kinds of food-borne pathogens in artificially contaminated food samples.Methods Target bacteria and non-target bacteria was inoculated on the blood agar plates and cultured at 36℃overnight.The concentration of the target bacteria was adjusted to 0.5 McF to make 10 fold serial dilutions.Then the DNA was extracted from dilutions to determine the sensitivity while the DNA of non-target bacteria was extracted directly to determine the specificity of the kits.Finally the kits were used to detect the 3 kinds of target bacterias in the food samples cultured according to the national standards of China after artificially contaminated by high,medium,and low concentrations of the target bacteria.Results The minimum detection limits of the kits for V.parahaemolyticus,E.coli O157:H7 and L.monocytogenes were 140,380 and 7900 CFU/mL,respectively,in culture medium,and 1.2 CFU/25 g,5.1 CFU/25 g,and 10 CFU/25 g,respectively,in artificially contaminated food samples.Only target strain was tested positive while non-target strains were tested negative for each kit.Conclusion The freeze-dried real-time PCR detection kits has good sensitivity and specificity,which is suitable for the detection of V.parahaemolyticus,E.coli O157:H7 and L.monocytogenes in food.