The Establishment Of Trace-rapid Field Detection Technology Of Six Pathogenic Microorganisms And The Development Of Groups Of Detection Cases

The economical development of china has gradually linked with the world today, it in high speed brought us many convenient while there are also a series problem for instance biosafety, the influence of it is not allow to neglect. In recent years, various virus give rise to virous infectious disease such us FMD、ASF、SF、ND、brucellosis、E.coli O157:H7 etc, it pose a serious threaten to public health security and cause a substantial damage as well. Testing pathogeny above major depends on serological diagnosis at present in our country and the low specificity and sensibility is still a problem. The etiology diagnosis that in different way and practical way is few especially in spot rapid diagnosis.This study aimed at the optimization and evaluation of the detection method,selecting the instruments can be used for on-site detection,Apparatus-packing and instrument-studying that providing quick、convenient and effective technology for rapid detection including Sample processing case、Rapid detection case of nucleic acid and Rapid detection case of immunology could help many kinds of method such as PCR、ELISA and collaurum.Firstly, checking literature of Chinese and English that related with six kinds of pathogenic microorganism above, and then select them by the best representative primer, Choose three different Taq enzyme:Es Taq Master Mix enzyme、Ahram High-speed enzyme、Takara Ex Taq enzyme. Program setting and parameters of machine are different from routine laboratory instruments for using miniaturized instruments that used in the experiment, so it is necessary to explore the experimental conditions, resuscitating、culturing and counting brucella melitensis M5、brucella abortus 104M、brucella suis S2、E.coli O157:H7、E.coli、Staphylococcus aureus、yersinia、Listeria monocytogenes、acinetobacter baumannii、Streptococcus suis type 2、Glycerol Salmonella bacteria that we preserved before, determining the titre of New castle disease、foot and mouth disease、CSFV after amplification that cultured by cells, amplificating plasmid of ASF after conversion.Secondly, Reverse transcription RT-PCR empirical method was created by extract related RNA of New castle disease virus、foot and mouth disease virus、CSFV that we used before; PCR was established after extracting plasmid of CSFV; PCR was established by extract related DNA of brucella and E.coli O157:H7 that cultured and propagated. These methods was evaluated from three aspect(specificity、sensibility and repeatability). Taking this way to propagate corresponding DNA sequence and then running agarose gel electrophoresis, according to the result to initial analysis by using Gel Imaging System. target fragment was extracted from gel and catenated to PMD-18 for sequencing, the similarity of the effect of sequencing was up to 99%.Finally,ELISA kit was employed to detect the artificial contaminated sample of E.coli O157:H7 and CSFV and compared with PCR(Taking the PCR test results as a "gold standard”),we can make a conclusion that this kind of ELISA kit is able to detect the corresponding pathogeny and achieves very good results.