The Pathogens Contamination Analysis Of Sold Cold Dishes And Its Rapid Detection Of PCR

Listeria monocytogenes,Staphylococcus aureus,Escherichia coil 0157,Salmonella spp.and Bacillus cereus are major food-borne pathogens that often contaminate food and cause a outbreak.Cold dishes make up a lot of traditional food in China.It is convenient for consumers as cold dishes can be eaten directly after purchase.However,cold dishes are susceptible to contamination by a variety of pathogens or spoilage bacteria during preparation,storage,transportation and sale.Lack of high temperature sterilization before eating cold dishes can result in food-borne illness if it is contaminated by pathogens.Conventional culture methods rely on isolation culture media,differential mediums and biochemical kits.The detection of pathogens can be time-consuming using conventional methods including non-selective and selective enrichment,differential selective plating and biochemical confirmation.There are many accurate and effective methods to detect the presence of single pathogen in food samples.However,when it comes to detecting several pathogens in one food,the existing methods are labor-intensive,expensive,complicated and time-consuming.Quick and easy Polymerase Chain Reaction(PCR)detection would significantly decrease the resources required in routine laboratory operations,and would enhance the overall efficiency of detection in food supervision and inspection.Moreover,multiplex PCR can detect more than two pathogens in one tube and would be quicker,easier and cheaper to run.The aim of this study was(1)to carry out a market survey using conventional culture methods to investigate the contamination of five food-borne pathogens in commercial cold dishes;(2)to establish a multiplex PCR assay based on novel molecular targets to simultaneously detect three pathogens contaminated in cold dishes;(3)to evaluate the detection effect of the multiplex PCR assay by stability testing and practical sample testing.Here follows the main results.The results of market survey showed that S.aureus caused the most contamination,with its positive rate reaching 8.08%,followed by L.monocytogenes(6.06%)and Salmonella(2.02%).No B.cereus or E.coli O157 was detected in any of the 99 cold dish samples.Among the three sampling locations surveyed,mobile vendors suffered from the worst pathogen contamination compared with farmers markets and supermarkets.Pathogen contamination rates in meat products was much higher than in the other two types of cold dishes.To establish a multiplex PCR system for detecting three pathogens contaminated in cold dishes,specificity and sensitivity of single PCR was carried out which was aimed at detecting S.aureus,L.monocytogenes and Salmonella separately.Three primer sets were designed based on the target gene and exhibited 100%inclusivity for target strains,and 100%exclusivity for 20 other strains in the single PCR.Sensitivity with pure DNA in the single PCR showed that the primer sets produced predicted specific amplified fragments with a lower detection limit of 340.3 fg/?L(SC8),10.4 fg/?L(lm16)and 173.7 fg/?L(Sa5).After optimization of the best amplification conditions for the multiplex PCR,we evaluated specificity of multiplex PCR.26 DNA template combinations were randomly selected from 66 target strains including 10 L.monocytogenes,39 Salmonella spp.and 17 S.aureus without duplication.Three expected bands for Salmonella,S.aureus and L.monocytogenes were produced across 26 kinds of target strains combinations.When detecting only one or two target strains,the multiplex PCR gave one or two corresponding specific amplified fragments.20 non-target strains did not produce any bands.The detection limit for pure cell cultures of multiplex PCR was 103 cfu/mL.The results of stability testing including different storage temperatures,different freezing and thawing times,reproducibility test of the same batch,reproducibility test of different batches were satisfactory.Practical sample testing suggested that pathogen contamination rate was basically consistent with previous market research.The detection rate of the three pathogens by multiplex PCR assay and conventional culture method was essentially the same,although the data for S.aureus was slightly different.The results of stability testing and practical sample testing suggest that this multiplex PCR assay will be a useful technique for the detection of S.aureus,L.monocytogenes and Salmonella in cold dishes.