Construction Of Salmonella Whole Genome Database And Its Molecular Targets Mining And Application Research

Salmonella is one of the most common foodborne pathogens,serotyping is an important basis for the harmfulness determination and clinical treatment of this bacterium.According to the difference of antigen structure,this genus can be divided into more than 50 serogroups and 2600 serotypes.The complex and diverse serotypes increase the difficulty of traditional detection and control methods.Molecular biological detection based on nucleic acid has become one of the most promising detection techniques to replace traditional detection methods,because of its characteristics of rapid,accurate and simple.The specificity and number of molecular targets are the bottleneck for the rapid detection of Salmonella serotyping.There is no systematic genomics study for foodborne Salmonella and the genomic data of various serotypes are not fully explored.Firstly,based on the preconstructed national food microbiological risk identification database and strain resource database,next generation sequencing and bioinformatics method were used to comprehensively analyze the foodborne Salmonella genome and construct the Salmonella whole genome database.Based on this,using pan-genome analysis and PCR verification,we systematically excavated new specific detection targets for Salmonella genera,serogroups and serotypes,corresponding with the establishment of rapid detection systems.Functional prediction,gene knockout,functional verification and transcriptomics were used to explore the biological functions and metabolic regulatory networks of Salmonella genera specific target genes.A novel phage with different Salmonella serotypes was screened from environmental soil and preliminarily applied to the prevention and control of Salmonella contamination in food by using biological characteristics and whole genome analysis.The main contents and results of them are as follows:(1)Construction of a Salmonella whole genome databaseA totoal of 351 Salmonella strains isolated from the national food samples in our group were sequencing,involving 8 serogroups and 44 serotypes.The genomic characteristics were analyzed comprehensively for better understand,including pan-genome analysis,genetic evolution analysis,virulence and drug-resistance gene.Studies have shown that Salmonella was a closed pan-genome with 29,241 genes and 1418 core genes,and they were clustering among different serotypes.Most of the strains carried virulence island-1 and virulence island-2 related genes,and the virulence genes were very similar among different serogroups.Serogroup B and serotypes London and Meleagridis in serogroup E carried more drug resistance genes,serogroup F,M and Q carried less drug resistance genes,and different serotypes within serogroups carried similar drug resistance genes.(2)Salmonella genus targets and loop mediated isothermal amplification(LAMP)detection methodThirteen molecular targets were identified as specific to Salmonella[3 molecular targets(mla B,group?25736 and group?28248 genes)were identified as specific to Salmonella genus including S.bongori and the remaining 10 molecular targets(ssa Q,yhh A,ssa H,ssa P,cit X?2,ssa O,ssc B,ssa S,ssa G and ssa T genes)were identified as specific to S.enterica and one target gene ssa Q was used in LAMP.The detection limit was 2.1×10¹CFU/m L and the sensitivity was high,which could realize the rapid identification of Salmonella in food samples.(3)Salmonella serogroup targets and multiplex polymerase chain reaction(m PCR)detection methodB-rfb J,C1-9679,C2-pim B,D-rfb J and E-28255 were identified as specific target genes for Salmonella serogroup B,C1,C2,D,and E,respectively.Three m PCR were constructed by combined with the targets of serovar Enteritidis and Typhimurium,which the common serogroups and serotypes can be detected simultaneously,and it saves detection time and cost.(4)Salmonella serotype targets and quantum dot nanobead-labelled lateral flow immunoassay strip based on strand displacement loop-mediated isothermal amplificationSixty-four new targets of 15 Salmonella serotypes were excavated,including serotype Typhimurium,Derby,Indiana and Agona in serogroup B,serotype Enteritidis in serogroup D,serotype Rissen,Braenderup,Infantis and Virchow in serogroup C1,serotype Hadar and Albany in serogroup C2,and serotype Weltevreden,London,Meleagridis and Senftenberg in serogroup E.The target has good specificity and high sensitivity,and can be successfully used in the process of food sample detection.Based on the S.Typhimurium target,quantum dot nanobead-labelled lateral flow immunoassay strip based on strand displacement loop-mediated isothermal amplification was established.Its specificity was good,with a naked LOD of 10³CFU/m L,which was 10 times more sensitive than Au NPs-labeled lateral flow immunoassay strip.It has a good linear relationship between 10²-10⁷CFU/m L,R²is0.969,and the LOD calculated by theory is 10^-1CFU/m L.The naked LOD in drinking water,orange juice,lettuce and chicken was 10³,10⁴,10⁴and 10⁵CFU/m L,respectively.The recoveries of the samples were in the range of 85%?110%,and the relative standard deviation was less than 7.8%.(5)Study on the function of Salmonella genus target related to virulence island-2 genesThe functions of most of the new target genes were predicted as putative proteins,among them,7 genera targets were related with virulence island-2 genes.The virulence island-2related gene ssa G,ssa H,ssa O,ssa P,ssa Q,ssa T and ssc B were knocked out.The results showed that the chromogenic reaction,growth performance and total protein expression did not change before and after knockout.There were some differences in forming biofilm ability.The cell adhesion(excepting?ssa Q),invasion,proliferation and toxicity of the knockout strains were reduced.It was found that most of the differentially expressed genes were related to the flagella,pili and ribosome synthesis,indicating that these genes were closely related to the motility and chemotaxis of bacteria.It is suggested that these 7 genes directly or indirectly affect the motility and chemotaxis of bacteria,so as to better exert its toxicity.(6)Isolation and application of a novel Salmonella phageFor the Salmonella different serotypes,a new phage v B?Sal P?TR2 was isolated.The phage was a short tail phage with high lytic potential against the serotype Albany,Corvallis,Newport,Kottbus and Istanbul.It possesses a certain tolerance against a range of p H and temperature,the incubation period of time is relatively short and it not carries any virulence or antibiotic resistance genes.In pure culture and food matrix,it has the ability to decrease a certain number of Salmonella.In summary,this study constructed the Salmonella whole genome database.Based this database,the experiments for the excavation of specific targets,the function of virulence island-2 related genes,and isolation and application of a novel phage were performed,which provide reference values for the rapid detection,the pathogenic mechanism,and effective prevention and control for Salmonella studies.