Molecular Serotyping Of Salmonella By Polymerase Chain Reaction

Objective Establish two methods for identifying Salmonella serotypes based on polymerase chain reaction.One way is to develop a gene pool composed of O and H antigen-encoding genes of Salmonella and design a software basing on the pool to determine Salmonella serotypes using O and H antigen-specific amplification sequences.The software can simultaneneously display serotypes of Salmonella,types of O-groups and H-antigens,and sequence identity.The other way is to establish a polymerase chain reaction method to rapidly detect Salmonella Anatum and to evaluate its specificity,sensitivity and detection limit.Methods In the first way,the O and H antigen-specific genes were obtained by three approachs including PCR amplification of 102 isolates covering 71 serotypes of Salmonella,literature review,and downloading corresponding sequences from genomes of Salmonella in GenBank database updated in September,2017.Then using R language codes,Mega 6.06 and other online software to analysis the sequences,and ultimately established a relatively complete O and H antigen gene-library.With this library,we edit a code using Python language to establish a software that can predict the serotypes of Salmonella.The accuracy of the predicting software was evaluated.In the other,the genomes of Salmonella spp.were downloaded from GenBank database and were blasted using bioinformatics’ tools to find certain specific genes which are only present in S.Anatum strains.Primers targeting the above specific genes of S.Anatum were designed and an assay of PCR was established.The specificity,sensitivity and detection limit of the assay were evaluated using pure cultured bacterial strains,clinical and simulated stool samples.Results Through the three approachs,we obtained 176 O-antigen sequences which represents 46 O antigens,and 1041 H-antigen sequences,representing 68 H antigens.The O-antigen sequences were clustered and analyzed using R language code.The sequences of different O antigens were significantly different;the H-antigen sequences were analyzed by using software,such as Mega 6.06.All H antigens were clearly divided into four clusters.With the exception of individual ones,the same antigens were all clustered on the same branches.Based on this,a relatively complete O and H-antigen gene-library was established followed by using Python language to write code and design a software.After the sequenced O and H-antigen gene from a Salmonella strain are deposited into the software,the serotype of the strain,O-group,H antigens and sequence identities are exported.The accuracy of the software is 99.39%for O-group,97.21%for H antigens and 70.6%for the serotypes.In the identification of S.Anatum,a PCR assay for rapid detection of S.Anatum based on a novel marker gene AW58 15605 was established.The specificity tests of the assay showed that AW58₁5605 could be amplified in all S.Anatum strains,whereas no amplification was observed in other serotypes of Salmonella strains and other enteric pathogens causing diarrheal diseases.The detection limit of the PCR assay for S.Anatum with purified DNA as template reached 59.1 cfu/g in simulated stool samples after overnight selective enrichment,which was 105 fold higher than that of pre-enrichment.Conclusion In this study,a relatively complete O and H-antigen gene-library was established and basing on that a software that can use the amplified sequences of O and H antigens to determine the serotypes of Salmonella is developed.And the established PCR assay for rapid detection of S.Anatum based on a novel gene could recognize S.Anatum with high specificity and sensitivity.Using this novel method and strategy,we can more easily,rapidly,and accurately to identify Salmonella than does the traditional method,making it as an alternative method to traditional approach.Moreover,it can be widely applied with the characterization of simple manipulation.And it has a good potential for application in Salmonella outbreak response.