| Salmonella belongs to Enterobacteriaceae bacteria,with more than 2600 serotypes,among which pullorum and typhoid are the main pathogens caused Salmonella infection in poultry.Salmonella is common in poultry and is mainly found in the intestinal tract.The complex serotype and high infection rate of Salmonella are very serious to the poultry industry.When Salmonella infection occurs in chicken flocks in intensive farms,effective antimicrobial therapy is still the most important control means.However,improper selection or abuse of antimicrobial drugs will not only reduce the efficacy of drug treatment,but also induce bacterial resistance.In order to control Salmonella infection,it is of great practical significance to identify and type Salmonella timely and accurately.The aim of this study was to establish a quadruplex PCR method for the identification of Salmonella serotypes,and to provide an effective tool for the accurate classification and identification of clinical Salmonella,which is of great significance for the prevention and control of Salmonella,Through the investigation of the prevalence and drug resistance of these four Salmonella serotypes in Shandong,it can provide reference for the prevalence of Salmonella and the rational use of antibiotics,so it has practical guiding significance for the scientific use of antibiotics.In this study,primer design was performed based on GenBank database,Bioedit and Primer 6 software,and the feasibility and specificity of the proposed method were verified by amplification of target genes of Salmonella and seven non-Salmonella strains.The target strains were cultured for enrichment,the OD value of the bacterial inoculum was calibrated to 108 CFU/mL,and then adjusted to 101 CFU/mL by 10-fold serial dilution.Subsequently,the established method was used to amplify the mixed templates of each gradient,and the minimum detection limit of the method was obtained.The established quadruplex PCR method was used to detect and identify Salmonella in poultry samples.Simultaneously,the samples were identified by 16s rDNA sequencing,and the results were compared with that of the established method to determine the accuracy of the quadruplex PCR method.The results showed that the primers designed in this study could effectively amplify the target genes,whereas the results of amplification of bacteria were negative.Sensitivity test showed a minimum bacterial concentration of 102 CFU/mL.There were 128 positive samples for Salmonella,of which Salmonella enterica was 39%,Salmonella pullorum was 11.7%,Salmonella gallinarum was 2.3%,Salmonella typhimurium was 33%and other serotypes were 14%by the quadruplex PCR.Furthermore,the results were consistent with the 16s rDNA sequencing.Overall,the established quadruplex PCR method for Salmonella identification is accurate,reliable,specific,sensitive and stable,which is expected to be an effective method for Salmonella monitoring in the future.Kirby-Bauer antibiotic testing was utilized to investigate the minimum inhibitory concentration of identified Salmonella to various antibiotics.The results showed that the resistance rates of Salmonella to amoxicillin,ceftazidime,ampicillin,trimethoprim,cefotaxime,tetracycline,Spectinomycin and ofloxacin were 45%,42%,40%,36%,33%,28%,15%and 8%respectively,while all of them were susceptible to enrofloxacin,florfenicol,gentamicin,meropenem and polymyxin.The results of the drug-resistant phenotype indicate that long-term use of antibiotics to treat salmonellosis has led to a decrease in the sensitivity of salmonella to some antibiotics and a certain degree of drug resistance.. |
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