Research Application On Specific Multiplex PCR Detection Methods Of Mycobacterium Bovis

Mycobacterium bovis is the causive agent of bovine tuberculosis in a range of animal species and human beings. The prevalence and circulation of this disease not only influence the development of stock raising severely, but also menace the health of human beings, so it was recognized as B infection by OIE. More than10%of human tuberculosis is caused by M. bovis. In recent years, the incidence of human tuberculosis has raised in large extents, the death toll has reached3millions every year, which is the accumulation of death toll of other infections, so it is the ringleader among all the infections. Because of the prevalence of bovine tuberculosis, human tuberculosis can not be eradicated. Recently, the incidence of bovine tuberculosis has also raised largely, especially in the developing countries. In our country, the positive incidence is about10%.Bovine tuberculosis is a kind of chronic and progressing disease, most cattles do not submit apparent infection or present clinical symptoms only in advanced stage, so accurate detection results are difficult to acquire in morning stage.Etiology is one of the valid detection methods, but this method present low positive ratio, so this detection method is not practical. Traditional detection of bovine tuberculosis is tardive allergy, which is the standard methods and the destine methods in international trade, this method often result in false positive and negative results.So this method is expensive,time-consuming,manpower and specifity-insufficient.。The triple PCR was used in this study to detect the cattle clinical samples.The detection results of triple PCR were compared with traditionary detection method. Base on above results, the triple PCR detection method can be identified wether it can be used in Clinical detection. In this study, the mainly study contents as follows.1.335cattle biood samples were detected with the triple PCR.2cattle whole blood showed positive results, the positive ratio was0.6%,the positive coincidence with ELISA reached100%. The specific genes were amplified by using the template of whole blood genomic DNA and specific primers based on the IS6110-IS1081-recA triple PCR system. Sequential and homogeneous analysis indicated that between M. bovis and other strains of M. bovis, the related specific genes are homogeneous in large extents.,the positive coincidence with ELISA reached100%. With the high positive coincidence, so triple PCR are suitable for the detection of M. bovis in molecular level. 2.360hilar lymph nodes were detected with the triple PCR.Only one lymph node sample showed positive result, the positive ratio was0.28%. All the hilar lymph nodes were processed and inoculated in improved-medium.These mediums were placed into37℃incubator and observed periodically.None of bacterial colony grow in all the medium.3. Two separate studies were undertaken on500cattle milk samples and the results were compared.These milk samples were detected by indirect ELISA and triple PCR.In the end, all results were negative.In a word, for the past many years, bovine tuberculosis prevailed generally because of the deficiency of efficient diagnostic methods. So the development of reliable and rapid screening tests have become the research hot. Polymerase chain reaction (PCR) methods offer great potential in this respect.