Molecular Rapid Detection Method Research Of Salmonella And Pathogenic Serotypes In Food

Salmonella is one of the most common foodborne pathogens,which often causes food poisoning around the world.Salmonella strains are divided into more than 2600 serovars that have the characteristics of the distribution of multi-regional.Therefore,rapid and reliable detection of Salmonella serotypes is essential.Conventional methods for detecting and identifying serotypes are laborious and time-consuming and cannot meet the requirements for rapid analysis of food companies and quality assurance departments.Development of a rapid and sensitive method for identification of Salmonella serotypes is desirable.In this study,several serotype-specific genes were identified by bio informatics and comparative genomics,included S.Enteritidis,S.Typhimurium,S.Paratyphi B,S.Paratypyi C,S.Dublin and S.Heidelberg.These serotype-specific genes were then used as target for rapid serotyping by multiplex PCR,real-time PCR and real-time NASBA.The main results of this study are as follows:1.Mining and evaluation of serotype-specific genes for the molecular detection of 6 Salmonella serotype.First,6 strains were considered to be representative of serotype,for example S.Typhimurium LT2?NC₀03197.1?,S.Enteritidis P125109?NC₀11294.1?,S.Paratyphi B SPB7?NC₀11205.1?,S.Paratypyi C RKS4594?NC₀12125.1?,S.Dublin CT₀2021853?NC₀11205.1?and S.Heidelberg SL476?NC₀11083.1?.These genes from every strain were aligned by the BLASTN program.Verified serotype-specific genes were only presented in genomes of strains in the same serotype but were not present in the genomes of the other serotype.As a result,8,12,18,7,15 and 10 verified serotype-specific genes were found for 6 serotypes,respectively.These genes were further validated by 48 Salmonella and 18 non-Salmonella.In this study,hsdS and STM4494 were serotype-specific genes of S.Typhimurium,kil?SEN1382 and SEN1383 were serotype-specific genes of S.Enteritidis,SPAB₀1122?SPAB 01123 and SPAB₀1124 were serotype-specific genes of S.Paratyphi B,SPC₀871?SPC₀872 and SPC₀908 were serotype-specific genes of S.Paratypyi C,SeD_A1118 and SeD_A2283 were serotype-specific genes of S.Dublin,SeHA_C2639?SeHA_C2640?SeHA_C325?SeHA_C3259 were serotype-specific genes of S.Heidelberg.The sensitivity of these primers targeting serotype-specific genes were 98.33ng/?l-140fg/?l According to the gene annotations,the 75%serotype-specific genes are associated with substance metabolism.The remaining part of the serotype-specific genes consist of phage genes,membrane protein genes and the genes of O-antigen synthesis.2.The detection of single Salmonella serotype by multiplex PCR assays.A multiplex PCR assay based on the SPAB 01124 and invA gene was developed and evaluated for detection of Salmonella spp.and S.Paratyphi B.The electrophoresis pattern only showed two bright specific bands at 284bp and 384bp in S.Paratyphi B without any non-specific bands.The specificity of the multiplex PCR reaction was evaluated by 48 Salmonella strains and 18 non-Salmonella strains.Meanwhile the multiplex PCR did not indicate any cross-reaction with 100%specificity.The detection limit was 1.91ng/ul and 2.2cfu/ml after 12h enrichment in pure culture.In artificially contaminated milk,the assay could detect less than 10cfu/10ml.And the S.Typhimurium and Escherichia coli did not interfere with PCR detection of S.Paratyphi B.A multiplex PCR assay based on the SeD_A1118?SeD_A2283 and invA gene was developed and evaluated for detection of Salmonella spp.and S.Dublin.The positive result only showed two bright specific bands at 284bp 378bp and 463bp in S.Dublin.The specificity of the multiplex PCR reaction was evaluated by 48 Salmonella strains and 18 non-Salmonella strains.Meanwhile the duplex PCR did not indicate any cross-reaction with 100%specificity.When temple concentration was less than 1.87pg/?l,the PCR assay only obtained two bands.Therefore,the detection limit was 1.87pg/?l.The sensitivity of the PCR assay was as few as 10 CFU/mL after 12 enrichment.It was shown that co-culture with S.Enteritidis and Escherichia coli up to concentrations of N×10³cfu/ml-N×10^-1 cfu/ml,respectively,did not interfere with PCR detection of S.Dublin.In artificially contaminated milk,the assay could detect less than 10cfu/10mlA multiplex PCR assay based on the SeHA_C2640?SeHA_C3259 and invA gene was developed and evaluated for detection of Salmonella spp.and S.Heidelberg.The positive result only showed two bright specific bands at 212bp?284bp and 589bp in S.Heidelberg.The specificity of the multiplex PCR reaction was evaluated by 48 Salmonella strains and 18 non-Salmonella strains.Meanwhile the multiplex PCR did not indicate any cross-reaction with 100%specificity.When DNA concentration was 1.4ng/?l,the PCR assay could obtained three bands?However,when temple concentration was 1.4fg/?l,the PCR assay only obtained two bands from 139-141 primer and pHAm9 primer.The sensitivity of the PCR assay was 3.8×10^-1 cfu/ml after 12 enrichment.Positive result were found for S.Heidelberg in the presence of up to 10⁴ cfu/ml of S.Typhimurium or Escherichia coli.In artificially contaminated milk,the assay could detect 3.8cfu/10ml after 10h enrichment.3.The detection of three Salmonella serotypes by multiplex PCR assays.A multiplex PCR assay based on the SEN1383?STM4494?SPC 0872 and invA gene was developed and evaluated for simultaneous detection of Salmonella spp.S.Enteritidis,S.Typhimurium and S.Paratyphi C.The positive result only showed four specific bands at 141bp?534bp?212bp and 284bp.The specificity of the multiplex PCR reaction was evaluated by 48 Salmonella strains and 18 non-Salmonella strains.Meanwhile the multiplex PCR did not indicate any cross-reaction with 100%specificity.The detection limit of 3 serotypes were less than 10cfu/ml after 10h enrichment in pure culture.In addition,it did not interfere with multiplex PCR detection of 3 serotype in the presence of up to 10⁴ cfu/ml of S.Dublin,S.Agona or S.Choleraesuis.In artificially contaminated milk,The detection limit of 3 serotypes were less than 10cfu/ml after 12h enrichment.4.Development and evaluation of real-time PCR assays for the detection of S.Heidelberg.A real-time PCR assay were developed for the detection of Salmonella and S.Heidelberg by amplifying SeHA_C3258 and invA genes.The result of real-time PCR was positive,when the two Ct values for the S.Heidelberg-specific JOE?SeHA_C3259?and the Salmonella spp.-specific FAM?invA?were less than 35 cycles The assay showed 100%specificity on all strains tested.The detection limit was 112.4fg/?l.The detection limit of the real-time PCR for pure cultures of S.Heidelberg was approximately 2.4×10⁴cfu/ml without enrichment step and 2.4cfu/ml after 12 h enrichment.In the presence of natural background flora enriched from chicken and pork breast samples,the detection limits were as few as 2.54×10³cfu/ml?1.7×10²cfu/ml.Therefore,the real-time PCR were nont affected by the presence of natural background flora of chicken and pork.The coefficients of variation of the assay were between 0.43-1.15%by amplifying different concentration of sample.A DNA extraction kit was more efficient at harvesting genomic DNA and reducing interference by PCR inhibitors.In artificially contaminated chicken and pork,the detection limits were lcfu/25g after 6h enrichment.5.Development and evaluation of real-time NASBA assays for the detection of Salmonella spp and S.Paratyphi CA real-time NASAB assay were developed for the detection of Salmonella by amplifying xcd genes.The assay showed 100%specificity on all strains tested.The detection limit was 10.5fg/?l of RNA.The detection limits of the real-time NAS BA were 1 CFU/ml of Salmonella spp after 10 h incubation in enrichment LB.The standard deviations of the assay were between 0.613-2.203 by amplifying different sample.When the samples were treated by DNase I and RNase A,the result showed that NASBA only amplified RNA.The results of the real-time NASBA and traditional microbiological methods were negative that the samples were 24 after heat treatment.In addition,the detection limit of the assay was 9.5×10³ cfu/ml in the presence of 4.75×107cfu/ml of natural background flora of pork.In artificially contaminated milk?beer and pork,the detection limits were less than 10cfu/25g after 10h enrichment.A real-time NASBA assay based on the xcd and SPC 0908 gene was developed and evaluated for simultaneous detection of Salmonella spp.?FAM?and S.Paratyphi C?JOE?.The positive result only showed the two Ct values.The real-time NASBA assay showed 100%specificity across 48 Salmonella strains?representing 34 serotypes?and 18 non-Salmonella strains.When RNA concentration was 40.94fg/ul,the assay could obtained two Ct values.The sensitivity of real-time NASBA system for pure culture and artificially contaminated chicken and pork was less than 10cfu/ml?25g?after 10h enrichment.In addition,the standard deviations of the assay were between 1.229-5.28 by amplifying different concentrations of sample.The detection limit of the assay was 10³ cfu/ml in the presence of 10⁸cfu/ml and 10⁷cfu/ml of natural background flora of pork and chicken.