Studies On Rapid Identification Method For Salmonella In Food And Feedstuff And B.anthrax In Animal Hair And Wool

Traditional bacterial classification methods depend on morphological character andbiochemical assay. Pure culture followed by morphological, biochemical and serologicalassays involved in the whole process. While in clinical cases, false positive results alwaysoccurred until the last step, biochemical and serological identification. Moreover, there aremany bacteria which can not be identified by VITEK, BIOLOG, and fatty acid assay system.In the process of Salmonella inspection, microbes which resemble Salmonella wereisolated on selective media. These non-target microbes always take troulbes to the followingidentification and waste much consumable materials, labour force and delayed inspcetion timeas well. Biochemical identification by instuments such as VITEK2, BIOLOG andPHOENIX-100, and serological identification would be carried out for suspect isolatedSalmonella.Biochemical identification is characterized by high-cost, long inspection cycleand low specificity. False positive results were occurred in serological assay which itself is atime wasting and labor exhausting. So, modification and development of new mothod isurgent to make up the deficiency of traditional method.In the process of inspection of B. anthrax, false-positive results always occurred. Somefamiliar bacillus which include B. thuringiensis, B. sphaericus, B. circulans, and B. Cereus,caused this result. So, more accurate and fast method was needed to be developed forinspcetion of B. anthrax.There are two goals in this research. Study on improved inspection method ofSalmonella based on the application of antisera against interference bacteria is the first goal.The targets were Proteus mirabilis and P. aeruginosa which were familiar in Salmonellainspection and cuase false positive results in our laboratory were used as research targets.Poly clonal antibodies against the two microbes was prepared through inoculating NewZealand rabbits respectively. Fast and accurate identification method for Salmonnella wasdeveloped through antimicrobial assay in vitro and clinical application. Secondly, PCR-RFLPwas applied to bacterial identification based on16S rDNA. PCR was used to amplify the pureculture and followed by enzyme digestion by17endonucleases respectively. Finger print ofRFLP was shown by agarose gel electrophoresis. The method developed could identify B.anthrax accurately and fastly. The research results were: (1) The sera of New Zealand rabbits which were inoculated by P. aeruginosa werecolleted. The purified IgG could inhibit and kill P. aeruginosa in vitro. The microbicidalactividy was confirmed by scanning eletron microscopy(SEM) and bacterial culture assay, i.e,P. aeruginosa became swolen and began to lyse when antibody of P. aeruginosa was addedinto broth in10minutes and lysed absolutely in80minutes. There was no bacterial growthfor lysate by culture assay. While, as control, E. cloacae was not affected by the antibody. Atthe same time, the prepared poly clonal antibody against P. aeruginosa could not inhibitSalmonella which was added into RVS broth and MkTTn broth.(2) The poly clonal antibody against P. aeruginosa was introduced into the process ofSalmonella, i.e. was added into the selective enrichment media. The growth of P. aeruginosawas inhibited obviously and so, P. aeruginosa was excluded in the process of Salmonella. Inclinical application, the rate of P. aeruginosa was19.8%（45/227）for Salmonella negativesamples with normal method. While, with modified method, the rate was only2.2%(5/227)which the accurate rate for Salmonella increased by89.2%. So, the introduction of antibodyagainst P. aeruginosa increased the accuracy of Salmonella isolation obviously coupled withthe decrease of cost and time of inspection.(3) The sera of New Zealand rabbits which were inoculated by Proteus mirabilis werecolleted. The purified IgG could inhibit and kill Proteus mirabilis in vitro. The activity wasalso effective to other strains of Proteus mirabilis. The microbicidal actividy was confirmedby scanning eletron microscopy(SEM) and bacterial culture assay, i.e, the obvious networkwas formed when Proteus mirabilis was treated by the antibody in2hours. The cultureassay showed that there was no bacterial growth for lysate. While, the prepared poly clonalantibody against Proteus mirabilis could not inhibit Salmonella and other enteric bacteria.(4) The poly clonal antibody against Proteus mirabilis was introduced into the processof Salmonella, i.e. was added into the selective enrichment media, SC and MM. The growthof Proteus mirabilis was inhibited obviously and so, Proteus mirabilis was excluded in theprocess of Salmonella. In clinical application, the false positive rate of Salmonella was57%(12/21) with normal method. While, with modified method, the rate was only18%(2/21)which the accurate rate for Salmonella increased by68.4%. So, as a specific inhibitant, theintroduction of antibody against P. aeruginosa increased the accuracy of Salmonella isolationobviously coupled with the decrease of cost and time of inspection.(5) Based on bacterial16S rDNA, method of finger print of PCR-RFLP was developed,i.e.17endonucleases were adopted to cut the amplificants of16S rDNA of bacterium andthen argarose electrophoresis was used to analyse the products. The results showed thatdeveloped method could identify the bacteria which lied in animal hair and wool fastly and accurately in48hours. At the same time, developed method could classify B. anthrax fromother baceria, escipecially bacillus. So, developed method could be an alternative method forB. anthrax identification.In conclusion, specific antibody which act as inhibitor was introduced into the process ofSalmonella isolation first time. The interference caused by relative bacteria was excludedsuccessfully. The application of specific antibody increased the accuracy of Salmonella inclinical samples obviously. So, this could save much money and labour force. At the sametime, finger print of PCR-RFLP based on bacterial16S rDNA was developed successfully.The method could identify bacteria isolated from animal hair and wool accurately withoutDNA sequencing. So, this assay could be applied to B. anthrax identification as an alternativemethod.