A Study On Nucleic Acid Detection Of Bacillus Cereus And Listeria Monoctyogenes

Bacillus cereus is widely found in soil,water,air and animal intestines,and it is also a major source of pollution for plant,animal and processed foods.Food poisoning can result from consumption of foods contaminated with Bacillus cereus,the most common symptoms of food poisoning are diarrhoea and vomiting.Diarrhea food poisoning is caused by a variety of heat-intolerant enterotoxins,while vomiting food poisoning is caused by vomiting toxin Cereulide.The traditional methods of Bacillus cereus isolation and culture have to go through the steps of enrichment,isolation and culture,microscopic examination,biochemical identification,etc.,which are time-consuming and cumbersome,and cannot identify whether they produce enterotoxin or vomiting toxin.There are now a variety of commercial kits for the detection of Bacillus cereus enterotoxin,but few rapid detection reagents for vomiting toxin.In this study,ET-PCR and MCDA techniques were used to establish a rapid detection method for emetic toxin producing Bacillus cereus.ET-PCR primers were designed with ces gene of Bacillus cereulide and 16S rDNA gene as detection targets,the specificity of the primers was verified by Blast comparison and nucleic acid amplification.The LOD of ET-PCR was evaluated and applied to the detection of artificially contaminated food samples.The established ET-PCR method for the detection of emetic toxin producing Bacillus cereus can achieve multiple detection of Bacillus cereus group and the emetic toxin gene at the same time,and the LOD was 100fg per reaction.The artificially contaminated food samples were cultured for 6h,and the LOD was 6.3 cfu/25g by ET-PCR method.At the same time,MCDA technology targeting on the ces gene,was applied to establish a more rapid,sensitive,specific and easy to operate method for the detection of emetic toxin producing Bacillus cereus.The best reaction temperature of MCDA method is 64?,the assay could be finished within 1 hour and the LOD is 100fg per reaction.After 6 hours of enrichment,the LOD for the simulated food samples was 6.3 cfu/25g using the MCDA method.The rapid detection methods of ET-PCR and MCDA for emetic toxin producing Bacillus cereus established in this study are specific,rapid,sensitive and easy to operate,which could be used for rapid screening of emetic toxin producing Bacillus cereus in food samples.Listeria monocytogenes is an important food-borne pathogen.Listeriosis of people is an infection caused by eating foods contaminated with Listeria monocytogenes,newborns,the elderly,pregnant women and people with low immunity are susceptible groups.The main clinical symptoms include sepsis,meningitis,pregnant woman abortion,the fatality rate of listeriosis was high to 20%-30%.The traditional isolation and culture method of Listeria monocytogenes commonly used in the laboratory requires twice enrichment,selective medium separation,preliminary screening and biochemical identification,the whole process takes 5-7 days.In recent years,many molecular diagnostic methods have been applied to the detection of Listeria monocytogenes,including traditional PCR method,multiple PCR method,real-time PCR method,LAMP method and MCDA method.According to the references,four nucleic acid methods based on the specific gene lmo0733 of L.monocytogenes were used for detection of the pathogen in the artificially contaminated samples and the raw meat samples.The reaction rate,detection sensitivity and detection specificity of four methods were compared.Our data indicated that the four nucleic acid methods could accurately detect L.monocytogenes in pure nucleic acid,artificially contaminated samples and raw pork meat samples.The whole process of MCDA assay could be completed within 30 minutes and the LOD of MCDA technique was 10fg of DNA template per reaction,the reaction rate and sensitivity of MCDA method are better than that of the other three methods of nucleic acid diagnosis.MCDA assay could be used as a rapid,sensitive and efficient method for detection of Listeria monocytogenes in meat product.And it could improve the positive detection rate of Listeria monocytogenes.