Establishment And Application Of LAMP Rapid Detection Method For Four Food-borne Pathogens

Food-borne pathogens are an important source of food safety problems.At present,the standard detection methods of foodborne pathogens are mainly the traditional methods such as isolation culture method or PCR nucleic acid amplification.These methods are time-consuming,complicated detection procedure,high detection cost and few detection targets,and rely on laboratory or large-scale equipment,it is difficult to meet the field of food-borne pathogens,low-cost,rapid detection requirements.In this study,loop-mediated isothermal amplification(LAMP)was used to detect four food-borne pathogens,namely,Vibrio parahaemolyticus,Listeria monocytogene,Enterobacter cloacae and Aspergillus fumigatus,the reaction system was successfully established,the primer sets corresponding to the four pathogenic bacteria were screened,and the specificity and sensitivity of the selected primers were tested.Finally,the established detection method was applied to real samples,this study demonstrated that LAMP technique can be used for rapid detection of food-borne pathogenic microorganisms.The main research findings and findings are as follows:The loop-mediated isothermal amplification was used to establish the bacterial LAMP reaction system and to screen the primers.The detection conditions were optimized and the sensitivity of the system was compared with PCR and QPCR,the results show that,the three pathogenic bacteria Vibrio parahaemolyticus tlH primers,Listeria monocytogenes plcB primers,and Enterobacter cloacae CigR primers were successfully screened,and the optimal reaction temperature for the three bacteria was obtained,with the reaction temperature at 65℃.The starting amplification time for all three bacteria was the earliest,and the specific amplification time could be set according to the detection limit.Betaine was tested at the optimal reaction temperature tested,and the optimal betaine reaction concentration for the three bacteria differed,with an optimal betaine concentration of 0.5 M for Vibrio parahaemolyticus,0.4 M for Listeria monocytogenes plcB priming,and 0.5 M for Enterobacter cloacae CigR priming.The primers for Vibrio parahaemolyticus tlH,Listeria monocytogenes plcB,Enterobacter cloacae CigR,and Aspergillus fumigatus β-tub were validated with good primer specificity.The detection limits were 1.78 × 102 copies μL-1 for Vibrio parahaemolyticus,3.06 × 102 copies μL-1 for Listeria monocytogenes and 1.8 × 101 copies μL-1 for Enterobacter cloacae,and compared with PCR and QPCR,the results showed that the LAMP assay was less sensitive and the amplification time was shorter,and the assay could be completed within The detection limit was compared with PCR and QPCR.The loop-mediated isothermal amplification was used to construct the detection system of Aspergillus fumigatus.The conditions were optimized,and the sensitivity of PCR was compared with that of QPCR,the results show that,It was verified that the designed β-tub gene is a set of high quality primers for Aspergillus fumigatus,which can only produce positive amplification for Aspergillus fumigatus,the amplification start time is less than 20 minutes,the optimal reaction temperature of LAMP reaction is 65℃,the optimal concentration of betaine in the reaction is 0.5 M,and the whole reaction can be finished detection within 45 minutes.The detection limits of LAMP in the genome of Aspergillus fumigatus extracted from pure bacterial solution were 3 × 100 copies/reaction,3 ×102 copies/reaction by PCR and 3 × 101 copies/reaction by QPCR,and the sensitivity of LAMP detection was lower than the other two.At the same time,we use HNB as the reaction visualization color change indicator,can clearly observe the reaction system kind of solution color change from violet to sky blue,assist in judging the results of the test,in the practical application kind this phenomenon to simplify the reaction steps,reduce the dependence on the instrumentation.The loop-mediated isothermal amplification(loop-mediated isothermal amplification)was used to detect the minimum detection limit of artificial contaminated samples,In the nucleic acid extraction of the simulated samples,some nucleic acids of the food matrix were also extracted,and in the specificity test of the simulated food matrix samples,we could make a preliminary judgment that LAMP is well tolerated by the food matrix,and the results of the detection limit of the simulated samples are similar to those of the single colony samples,which proves that the results are true and credible,and the accuracy test further proves that the primers we screened are high-quality primers for their target strains,and lays the foundation for the application of real food sample detection.