Mining Of Molecular Serotyping Targets Of Salmonella Isolates And Establishment Of Their PCR Method

Salmonella is one of the main pathogenic bacteria,which causes the highest cases of food poisoning,and it is a serious threat to human health.Rapid and accurate detection of Salmonella is the primary task of its prevention and control.At present,there are as many as 2610 serotypes of Salmonella isolated from the world,and there are about 300 serotypes in our country.These complex serotypes are important indicators for its detection.Therefore,development of fast and accurate Salmonella detection and serotyping methods is important for the prevention and control of its outbreak.Our previous studies have found that some specific detection targets obtained with Salmonella were in conservative and single nucleotide polymorphism(SNPs).The SNPs in different serotypes of 7Salmonella-specific detection targets were analyzed based on the whole genome sequence of 28 Salmonella strains(14 serotypes)downloaded from NCBI.The results in this study showed that S9 and S69 had more SNPs than others,which had the potentiality for the Salmonella serotyping.Subsequently,nucleotide base site difference table of the two candidate molecular serotyping targets were drawn by sorting out the differences of S9 and S69 in the Salmonella strains of different serotypes,respectively.Differential site combination of two target points for rapid molecular serotyping of 14 Salmonella serotypes cshowed that identical mutation sites existed in the same serovar,while different mutation sites were found in diverse serovars,the potential of molecular serotyping of S9 and S69 was further verified.Subsequently,this study focused on 38 serotypes of Salmonella in China,190 strains of Salmonella were amplified by PCR using moleculartargets of S9 and S69,and the amplified products were sequenced.Refer to the principle of clustering the housekeeping gene fragments in MLST,we mapped the SNPs differential loci of 2 molecular targets.The results of sequencing showed that the combination of these 2 molecular typing targets can differentiate the serotypes of the 38 Salmonella strains.At this point,we established the 38 common Salmonella serotype sites according to the table,this table can quickly identify the serotypes of Salmonella.In practical application,821 food samples were collected from supermarkets and open-air markets in Shagnhai,and 102 suspected Salmonella isolates were obtained.The results of molecular serotyping by targets S9 and S69 were consistent with traditional slide agglutination tests(coincidence rate 100%),including 7 different serovars: 35 strains of Salmonella enteritidis,28 strains of Salmonella typhimurium and Salmonella derby 14 strains,11 strains of Salmonella choleraesuis,8strains of Salmonella Newport,4 strains of Salmonella senftenberg,and 2strains of Salmonella Aberdeen.At this point,we established a one-step PCR method for Salmonella detection and serotyping.In summary,the results of genome sequence analysis and strain serotyping showed that combination of Salmonella-specific molecular detection targets S9 and S69 had the molecular serotyping capacity for Salmonella,which could be used in 38 types of serum typing,which can be used to replace the complex biochemical identification in the detection of Salmonella.And it is expected to be an alternative method for traditional of slide agglutination.It will reduce the time and cost based on the nucleotide base site difference table,which also provides new ideas for the application of PCR technology in Salmonella molecular serotyping.