Development Of A Mutiplex PCR For Detection Of Salmonella And Construction Of Salmonella Pullorum Aroa Mutants

Salmonella is a kind of intracellular pathogens that can results in a local and systemic infection. The infected hosts show the symptom of gastroenteritis, abortion, or septicemia. In some cases, the hosts show no symptom but as pathogen carriers. With wide use of antibiotics in clinic and animal feeds, more and more Salmonella with drug resistance are isolated and the rates of Salmonella infection in various animals are increasing, which results in huge economic losses. The tradition methods for identification of Salmonella include isolation, serotyping, and determination of biochemical characteristics, which is time-consuming and labor-consuming. In addition, it is difficult to differentiate some serotypes. Therefore, a rapid detection method of Salmonella spp. from avian species is definitely needed, and the effective prevent and control measures should be taken to reduce the infect rates of avian and to improve the quality of poultry products.In this study, five sets of primers were designed to amplify the genotype-specific gene segments of Salmonella spp.{hut), Salmonella enteritidisSdfⅠ), Salmonella typhimuriumiSpy), Salmonella pullorum{glgC) and Salmonella gallinarum(glgC、speC). The multiplex PCR method that could detect and identify Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum at the same time was established. Also, the deletion mutants of aroA gene including S004△aroA, S005△aroA, S017△aroA and S6702△aroA were constructed by λ Red homologous DNA technology, and their biological characteristics and virulence were detected to provide the evidence for the feasibility of the attenuated Salmonella vaccine in practice. 1The establishment of the multiplex PCR method for the simultaneous detection of Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarumFour primers were designed to amplify the serotype-specific gene of SdfⅠ (304bp), Spy(401bp), glgc(252bp), speC(174bp). The concentration of template DNA, the ratio of dNTP and DNA polymerase, and annealing temperature were adjusted, and a multiplex PCR method.was established. The result of sensitivity test of PCR amplification from bacterium solution showed that the minimum detectable limitation of Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum were all3×10CFU. The result of sensitivity of the target DNA showed that the minimum detectable limitation of Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum were162pg/μL,202pg/μL,214pg/μL,178pg/μL, respectively. The sensitivity, specificity and coincidence rate of the mPCR method for the simultaneous detection of Salmonella typhimurium were all100% compared to the routine bacterial culture and isolation. The sensitivity, specificity and coincidence rate of the mPCR method for the simultaneous detection of Salmonella enteritidis were100%,98%,98.2%, respectively. The sensitivity, specificity and coincidence rate of the mPCR method for the simultaneous detection of Salmonella pullorum were100%,94.6%,96.4%, respectively. The uninvolved serotype of Salmonella spp. clinical isolates could be detected by amplification of the genus-specific hut gene.2. The construction of the deletion mutant of aroA and the study of biological characteristicsThe aroA gene of S.pullorum clinical isolates was amplified according to the sequence deposited in NCBI. The deletion mutants of aroA including S004△aroA, S005△aroA, S017△aroA and S6702△aroA were constructed by λ Red homologous DNA technology, were confirmed by PCR identification.The growth curve of the four wild and mutant strains showed that S6702grew faster among the wild-type strains, and the other strains grew with similar speed. The mutant strains grew slower when compared with the wild-type strains, and S6702AaroA grew faster among the mutant strains. To determine the median lethal dose (LD50),2-day-old SPF chickens were inoculated intramuscularly with different dose of wild and mutant strains. The result showed that the LD5Os for wild-type S004, S005, S017, S6702were10630,107.040,106.612, and108.34, respectively. The LD50S for mutant strains S004△aroA, S005△aroA, S017△aroA, and S6402△aroA were108.60,108256,108.190, and10857, respectively. The data indicated that the virulence ofS004△aroA were significantly reduced, and the virulence of S6702AaroA had the minimum reduce while the virulence of wild S6702were lower than that of any other wild strains. Therefore, attenuation of S. pullorum by deletion of aroA gene depends on the strain. This study may lay a foundation for the screen of attenuated vaccine candidates.