Application Of RT-qPCR To Study The Expression Of Virulence Factors Of Listeria Monocytogenes In Simulated Systems

Listeria monocytogenes is one of the primary foodborne pathogens worldwide.Its resistance to the harsh gastric fluid plays a major role in the food poisoning of LM.In our present study,four representative strains were investigated including LM440?ATCC19115?ATCC19111and ATCC15313.All four tested strains were subjected to survey thetemperature?NaCl stress and the optimum pH range.Our results showed that four tested strains were harvested the highest OD600nm value during 8?12h cultured,meanwhile,all of them possessed the main virulence factors(hlyA,inlB,prfA and sigB)of LM and the optimum pH range was 7?8.Four strains had tolerance to high and low temperature,the lag time for growth was 45 ?<22?<4 ?<7?;the salt concentration range was 0-6%,and ATCC19115 can growslowlyin 8%salt;the pH range was 5-9.5 and LM440 is significantly higher than the other strains(p<0.05),when the pH = 5;Moreover,when the value of pH was 3.5,the highest survival rate came from LM440 followed by ATCC19115,ATCC15313 and ATCC19111;as pH 2.5,the ability to resist to the simulating human stomach of LM440 and ATCC19115 were significantly stronger than ATCC15313 and ATCC19111(p<0.01);as pH = 1.5,four tested strains were death after 2 h,however,at 1 h,the ordering of survival rate was ATCC19115>LM440>ATCC19111>ATCC15313.Based on above results,the tolerance to the simulating human stomach of four tested strains was illuminated.Further,ATCC19115grown insimulated salmon matrix system and extracted the RNA;LM440 grown in simulated digestive system of stomach and RNA was extracted using the RNA Extraction Kit(TianGen,Dalian),then the expression level of of hlyA,inlB,prfA and sigB were assessed by the RT-qPCR.The RT-qPCR data showed that the expression level of all four major virulence genes were up-regulation in a salmon matrix compared to on the TSA-0.6%YE(Tryptone Soya Agar-0.6%Yeast Extrat).Among of these four genes,the expression of prfA and sigB ofLM from salmon matrix stored at 4? exhibited significantly higher than those at 0 and 7?(P<0.01).Stored for 24 h at different temperatures,the expression level of in1B and hlyA were changing within 7?>4?>0?(P<0.01,P<0.01,P<0.01),and in the salmon matrix,the expression level of all four major virulence genes at 48 h exhibited significantly higher than 24 h(P<0.01).LM 440 wereadded to artificial gastric fluid,in the high temperature,the LM440 had high survival rate,the expression level of of hlyA,inlB,prfA and sigB were some relativities with artificial gastric fluid?culture medium?temperature and duration.