The Clone Of Toxigenic Pasteurella Multocida ToxA Gene And The Establishment Of PCR Diagnosis Method

The Pasteurella multocida (PM) distributes widely in the world, the rate of this bacteria carried on the animal and poultry is so high, some datas pointed out that this rate was 30.9% in the nose depth and larynx of the pig. Toxigenic Pasteurella multocida (T~+PM) can induce the withering rhinitis which is one of five big infectious deseases in the production on a large scale to raise pig and B degree animal infectious disease evaluated by OIE bureau. This disease was taken into our Country through the import of sire, and occured as defferent degreeand spread in many provinces and cities(districts) of china. Withthe improving of integrated degree, the harm taken by this disease became much serious for the pig-breeding industry. The disease incidence reached as high as 60% and induced the serious economy expense in China. T~+PM is the main germ of the Progressive atrophic rhinitis (PAR).To examine and separate T~+PM was the keypoint for diagnosis and co-ntroling this disease. Based on the character of this Pastruella multacidato- xin(PMT), many methods for exmamination of T~+PM were established, example the experiment of Cutaneous necrosis of the cavia cobaya, the experiment of causing death of the mouse, the experiment of promoting cell growth, the toxicity experiment of the fetus cattle lung cell ,ELTSA and so on. Regardless of animal experiment or cell culture, the T~+PM must be separated firstly, so operations were tediously, time-consuming and has a big limitation on clinic, but the PCR diagnosis method has many mer- its, example specificity, rapidity ,sensitivity and so on. The T~+PM needn't be separated,when using this method to diagnose the Toxigenic Pasturella multocuia simply, quickly and exactly.To analyse the homology of gene sequence of Toxigenic Pasturella multocuia that reported by Kamp in this experiment, and select the conservation sequence of Toxigenic Pasturella multocuia as a amplification region, we selected a pair of suitable PCR primer which amplification fragment is 338bp using Oligo 6.0 and the premier 5.0 software, this fragment was connected on the PGZM-T Easy carrier successfully and the recombination plasmid of specificity gene fragment of Toxigenic Pasturella multocuia was constructed. Through analysis of the recombination pl- asmid using the PCR attestation as well as the sequence analysis after analysis of the restrictive incision enzyme Not I enzyme ,we identified the gene cloniced was a specificity gene of Toxigenic Pasturella multocuia and established the PCR diagnosis method of Toxigenic Pasturella multocuia. The results showed the homolofy between the gene sequence of the standard roxigenic Pasturella multocuia and the specificical gene of Toxigenic Pasturella multocuia separated in local place was 97.94%. the result of the special and sensitive experiments indicated that we can't apply this sequence diagnosed to amplificate the golden yellow Staph, chain coccus, salmonella, Bacillus coli, septico- wave bacillus DNA; when the content of the Toxigenic Pasturella multocuia DNA was 432.6fg/ul, the clear stripbelt was still amplificated. To examine 55 swab samples from Yanbian area using PCR technology, the rate of picking out was 16.36%;the diagnosis of PCR established has many merits, example specificity, rapidity ,sensitivity and is a reliable diagnosis method for the disease of toxigenic Pasturella multocuia.