PMA-RF-SRCA检测乳中单增李斯特氏菌活菌

Detection of Live Listeria Monocytogenes in Milk by Real-time Fluorescence Saltatory Rolling Circle Amplification Combined with Propidium Monoazide

本研究建立了一种叠氮溴化丙锭(PMA)与实时荧光跨越式滚环等温扩增(RF-SRCA)相结合的方法,可以高效灵敏检测乳中活的单增李斯特氏菌。以hlyA基因为靶点,设计并筛选出特异性引物,通过对PMA的工作浓度、暗孵育时间和光反应时间进行优化,确定了最佳的处理条件。此外,对该方法的特异性、灵敏度及检出限进行了分析。结果表明,该方法引物特异性良好,6株单增李斯特氏菌结果均为阳性,12株非单增李斯特氏菌结果均为阴性;当PMA工作浓度为30μmol/L、暗孵育10 min、光反应15 min时,所建立的方法灵敏度为3.9 CFU/mL,人工污染乳制品的检出限为1.56×10 CFU/mL。综上所述,本研究所建立的PMA-RF-SRCA方法特异性强,灵敏度高,为检测食品中活的单增李斯特氏菌提供了新的思路。

In this study,a real-time fluorescent saltatory rolling circle amplification technology combined with propidium monoazide(PMA)was developed for efficient and sensitive detection of live Listeria monocytogenes in milk.Specific primers were designed and screened based on hlyA gene of Listeria monocytogenes.The working concentration of PMA,dark incubation time and light reaction time were optimized.In addition,the specificity,sensitivity and detection limit of the method were analyzed.The results showed that the specificity of the primers was excellent.Six Listeria monocytogenes strains presented positive results,while negative results were obtained from 12 non-Listeria monocytogenes strains.The optimum concentration of PMA is 30μmol/L.The optimum dark incubation time is 10 min,and the light reaction time is 15 min.The sensitivity of the established method is 3.9 CFU/mL,and the detection limit of artificially contaminated dairy products is 1.56×10 CFU/mL.In summary,the PMA-RF-SRCA method presented strong specificity and high sensitivity,providing a new idea for the detection of live Listeria monocytogenes in food.