Simultaneous Detection Of Foodborne Pathogens By High Resolution Melting Analysis

Foodborne diseases are widespread public health concerns.Many foods,such as milk, diaryproducts, meat, poultry, fruits, and vegetables, are commonly contaiminated with carriers ofmultiple pathogens such as Salmonella, L. monocytogenes,S. aureus and so on, methods thatcan detect several pathogens simultaneously were needed.To develope low-cost, rapid andspecific multiple systems for detection of Salmonella, L. monocytogenes and S. aureus, threepairs of specific primers were used targeting the invA hly and Sa442genes and two detectionsystems HRM-real time PCR and HRM-tHDA were set up.1The HRM-real time PCR method. A multiple HRM-real time PCR system wasestablished and optimized,based on the newly-built simplex HRM-real time PCR system.Themultiple system was not only evaluated by specificity, sensitivity and reproducibilityanalyses.but also applied to the artificially-inoculated and naturally-contaminated foodsamples. The multiple HRM-real time PCR system (25μL)was:10×buffer2.5μL,Taqpolymerase (5U/μL)0.5μL,Taq polymerase antibodies(5U/μL)0.5μL,dNTP(10mmol/L)2.5μL,MgCl2(25mmol/L)3.0μL,upstream/downstream primers(10μmol/L)0.5μL,template(0.5μmol/L)2.0μ,EvaGreen(20mg/mL)1.25μL BSA (20mg/mL)0.2μL, add ddH2O to25μL.Response procedures:95for10min,95for10s,60for45s40cycles. Meltingprocess:95for2min,60for2min,60for1s rise to95,refrigeration at50,melt rate at0.2/s.Specificity test showed that, there was no non-specific amplification of the44non-targetpathogens, whereas90target pathogens were detected and actual Tmvalues were79.4±0.14,82.5±0.15, and77.4±0.14for Salmonella, L. monocytogenes, and S. aureus, respectively.Sensitivity of the method in the single system for Salmonella,L. monocytogenes and S. aureuswere all102CFU/mL, respectively,and was102,102and103CFU/mL, respectively,in thesimultaneous detection.Reproducibility analyses showed that CVs of Tmvalues were all below2.17%in intra-assay, and all below3.93%in inter-assay. The detection limit inartificially-inoculated samples (n=50) was5CFU/25g food for all the3tested pathogens.In120naturally-contaminated pork samples,Salmonella DNA was detected by HRM, sequencing, and GB methods at a positive rate of25.00%,25.00%, and24.17%, respectively,thecorresponding rates for L. monocytogenes and S. aureus were all the same by three methods.2The HRM-tHDA method.A method to obtain crude Tte-uvrD helicase wasestablished.Gene tte-uvrd was cloned and Tte-uvrD was expressed.Crude enzyme obtained byultrasonication,centrifugation,sedimentation,dialysis and freeze-drying has a high tHDAdetection sensitivity and stability.A HRM-tHDA system was established.The system50μL was: crude Tte-uvrD helicase160ng/μL3.5μL,10×Annealing buffer5.0μL dNTP Mixture10mmol/L each3.5μL NaCl500mmol/L4.0μL MgSO4100mmol/L2.0μL,upstream/downstreamprimers10μmol/L1.0μL template DNA105CFU/mL2.0μL20×EvaGreen0.5μLddH2O add to50.0μL Response procedures:635s,62for55s,40cycles.Melting process:95for2min60for2min60for1s rise to95, refrigerationat50, melt rate at0.2/s.Specificity test showed that, there was no non-specific amplification of the30non-targetpathogens, whereas30target pathogens were detected and actual Tmvalues were79.4±0.1382.4±0.1177.5±0.16for Salmonella, L. monocytogenes, and S. aureus,respectively. Sensitivity of the system on the3tested pathogens were all102CFU/mL.Reproducibility analyses showed that CVs of Tmvalues were all below2.61%inintra-assay, and all below2.97%in inter-assay. The detection limits in50artificially-inoculated samples were all5CFU/25g food for the3tested pathogens.Detectionon50naturally-contaminated pork samples showed the same sensitivity with the GB method.HRM-real time PCR and HRM-tHDA detection systems were developed and optimized inthis study to reach high specificity,sensitive and repeatability. HRM-real time PCR system hasbeen applied to the foodborne pathogens multiplex detection. They are rapid,easy-conductedand low-cost. The development of new multiple detection system has a strong practicalapplication value and makes a great contribution on the food safety.