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Establishment Of Milk And Milk Products For Rapid Identification Of Mycobacterium Paratuberculosis Methods

Posted on:2016-10-28Degree:MasterType:Thesis
Country:ChinaCandidate:H X LiangFull Text:PDF
GTID:2283330470465362Subject:Prevention of Veterinary Medicine
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Paratuberculosis (Paratuberculosis) is the Mycobacterium paratuberculosis, which Mycobacterium avium subspecies paratuberculosis (Mycobacterium avium subsp.Paratuberculosis, Map) caused by a chronic wasting diseases, also known as Johne’s disease. The disease mainly infects ruminants, it is difficult purification, but also cause human Crohn’s (Crohn’s) disease, so early diagnosis of infected animals, has important implications for prevention and treatment of this disease.In this study, the detection method for the rapid identification of Map established using highly specific DNA probe technology, culture Mycobacterium paratuberculosis P10 and P18 both strains produced in a liquid medium, the genomic DNA, DNAStar software application for all gene fragment of M. paratuberculosis were compared to elect a consensus sequence specific gene and gene fragments of bacteria ISMAV2 application primer Express Software v2.0 software to design primers and probes were successfully constructed recombinant plasmids for TaqMan PCR standard curve established by the specificity of the test, to establish a TaqMan real-time PCR method, the experimental results of a rapid detection of Mycobacterium paratuberculosis show that the method is highly specific, and the success of this detection method to build on rapid detection and early diagnosis of milk and milk products Mycobacterium paratuberculosis has important significance.In order to establish a method for rapid differential diagnosis of Mycobacterium paratuberculosis live/dead cells. TaqMan real-time PCR method applied in this experiment with the establishment of PMA dye combination can only bind to DNA molecules according to PMA dead bacteria and inhibit amplification of dead bacteria principle, by optimizing the concentration of PMA and light reaction conditions, to determine the optimal reaction conditions, and optimization under good conditions, the application of PMA on milk samples in boiling water bath to inactivate Map processed by a TaqMan PCR detection of sterilized milk samples Map survival status. The results show:PMA at the concentration of 10μg/mL. BLU-V system illumination when the reaction time was 20min. PMA can be effectively combined with dead bacteria DNA. and does not affect the viable amplification:inactivation of milk samples was not detected Viable.
Keywords/Search Tags:Mycobacterium paratuberculosis, Fluorescence quantitative PCR, PMA, BLU-V system
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