Study On The Detaction Of Shigella Spp. Salmonella Spp. And Proteus Vulgaris By Multiplex PCR

Food safety emergencies are high frequency in recent years.In these problems,the food poisoning result of Bacterial Pathogens was the most harmful.According to data came from hospitals and inspection-quarantine departments.Rapid detection of food-borne bacteria is essential to efficiently prevent pathogens prevalence and food poisoning.The main bacterial pathogens were Staphylococcus aureus,Salmonella spp., Shigella spp.,listeria monocytogenes, Proteus vulgaris and so on. Since contamination levels are generally low in foods,detection methods require lengthy culture enrichment steps to increase target bacterial numbers before isolation and identification using standard cultural procedues.These conventional food microbiological techniques often require several days to detect bacterial pathogens present in foods at low levels.With the advent of PCR,Individual PCR assays have been developed for detection and identification of the bacterial pathogens.but a large number of individual PCR assays would be necessary if single primer sets are used in separate reactions on a large number of food samples,which can be a relatively costly and time- consuming process.To reduce the number of tests needed for diagnosis of Individual PCR assays,the simultaneous detection of several pathogens with a multiplex PCR (m-PCR) approach would be relatively rapid and cost-effective.So multiplex PCR which can simultaneously detect more than one bacterium has been applied increasingly conmprehensive.In this study,m-PCR assays were developed for the simultaneous detection of Shigella spp., Salmonella spp. and Proteus vulgaris,took use of Shigell .spp ipaH gene, Salmonella spp ipaB gene,and atpD gene of Proteus vulgaris,according to the result of BLAST to design three special primers.This m- PCR assay was developed for the simultaneous detection of Shigella spp,Salmonella spp and Proteus vulgaris from meat.In the PCR process,DNA concentration,Mg2+ concentration,template volume,annealing temperature and Taq enzyme concentration are optimized to determine the optimal PCR.The reaction mixture consisted of 25μL: 2.5μL10×PCR buffer,50 mMMg2+ 1μL,1μL mixture of dNTPs,1μL of 10μM forward primer(each),1μL of 10μM reverse primer(each),0.3μL (5 U/μL)Taq enzyme,ddH2O 11.5μL.The reaction was run under the following conditions:Cool start;DNA pre-denaturation at 95℃for 5min , DNA denaturation at 94℃for 45 s,primer annealing at 56℃for 45 s,and DNA extention at 72℃for 45 s,run 30 cycles;the final extention was performed at 72℃for 10min.The PCR products were examined by l.5% agarose gel electrophoresis.PCR products were confirmed by DNA sequencing.The result of sequencing compared with the target gene sequence at gene bank showed that PCR amplified products were certified.There were 15 bacterial strains to be detected including 2 Shigella spp,4 Salmonella spp,1 Proteus vulgaris,and 7 other bacterial strains in order to determine specificity of amplification of primers.The results of 2 Shigella spp, 4 Salmonella spp and 1 Proteus vulgaris were positive and those of 7 other strains were negative.The specificity of the m-PCR assay was evaluated by testing the three primer sets with the purified DNAs of all the strains(separately or in different combinations) indicated above.Positive PCR amplification of DNA templates from Shigellas app,Salmonella spp,and Proteus vulgaris produced a single fragment,of the expected,for each pathogen.The three bacterial pathogens were simultaneously amplified.The sensitivity of m-PCR for the detoction of one of three bacterial pathogens when using purified DNA of bacterial type strains was 102 CFU/ml for Shigella spp, 101 CFU/ml for Salmonella spp,and 102 CFU/ml for Proteus vulgaris.The sensitivity of the simultaneous detection of the three bacterial pathogens with m-PCR when using purified DNA of the bacterial type strains was 102 CFU/ml for Shigella spp, 102 CFU/ml for Salmonella spp,and 102 CFU/ml for Proteus vulgaris.The bacterial pathogens DNA was directly extracted using DNA KIT.The extracted DNA was suitable for m-PCR detection.The detection limit of the m- PCR assay for meat was102 CFU/ml for Shigella spp, 103 CFU/ml for Salmonella spp,and 102 CFU/ml for Proteus vulgaris.The whole expe rimental procedure can be completed only 6 hours,shorter than the traditional biochemistry detection.The developed method in this assay allows detection of the pathogens in meat in less than 6h.This method is useful for the detection of Listeria monocytogenes infood.