Study On The Plasmid Characters And PCR Identification Of Pasteurella Multocida

Pasteurella multocida is Gram-negative, non-motile coccobacillus that is penicillin-sensitive and belongs to the Pasteurellaceae family. It can cause a zoonotic infection in humans, which typically is a result of bites or scratches from domestic pets. Many mammals and fowl harbor it as part of their normal respiratory microbiota, displaying asymptomatic colonization. Therefor the prevention and treatment of pasteurellosis has always been the focus and difficulties of research at home and abroad. In this thesis, the PCR identify method of Pasteurella multocida had been studied with 36 strains of Pasteurella multocida as the material, and the plasmids of three strains of Pasteurella multocida had been studied with the methods of restriction digestion and Random Amplified Polymorphic DNA (RAPD) using the molecular biology techniques, obtained the following results:1. According to the gene sequence of Pasteurella multocida published in NCBI, a pair of specific primers was designed to amplify the DNA of 32 Pasteurella multocida, the detection rate reached 91%. In addition, the detection results of 5 other common pathogen bacteria were negative by using the pair of primers. This study demonstrated that this pair of primers are highly specific to Pasteurella multocida, and the PCR process could be used as a reliable method to diagnose the infection of Pasteurella.2. PCR test, biochemical assays and other methods were used to identify the chickens which were suspicious of infected by Pasteurella multocida, they were diagnosed to be infected by Pasteurella multocida indeed. Four wild strains of Pasteurella multocida were isolated. A DNA fragment of 1 226 bp was amplified and recovered, and the product of recovery was sequenced and compared with asparagine synthetase (ASN A) sequence of Pasteurella multocida subsp.Multocida str.Pm70 published in NCBI. Homology is 98%.3. The antibiotic sensitivity test of isolates showed that all strains of Pasteurella multocida were highly sensitive to the Pioneer cefotaxime in the 14 antibiotics. This provides an experimental basis for the treatment of the disease.4. The plasmids of Pasteurella multocida, which had been identified positive by PCR, were extract using alkaline lysis method. The result showed that rate plasmid-bearing is 47.4% and three strains (No. A, C, and 28) contain smaller plasmid.5. The three smaller plasmid were studied with restriction enzyme digestion and randomly amplification.The results showed that:plasmid of No. A contains no EcoRâ… recognition site, and two Hindâ…¢sites. No.C plasmid contains two EcoRâ… recognition site and one Hindâ…¢recognition site. No.28 plasmid contains at least one EcoRâ… recognition site and two Hindâ…¢recognition site.6. Three Pasteurella multocida plasmid (No. A, C, and 28) was amplified with 20 random primers. The results showed that:(1) Three to four DNA bands could be observed when amplifing the plasmid of Pasteurella multocida No. A with the random primers of No.17 and 18,and the bands were clear. (2) Two to three DNA bands could be observed when amplifing the plasmid of No.C with the random primers of No.5 and 13,and the bands were clear. (3) Three to five DNA bands could be observered when amplifing the plasmid of No.28 with the random primers of No.4 and 6, and the bands were cleare and bright. In addition, only two amplified bands could be observered with the primer No.14, but they were very clear and bright.