| P.is a gram-negative conditional pathogen capable of infecting a variety of hosts,including humans,companion animals,domestic animals and wildlife,and is a pathogen of avian cholera,bovine and buffalo hemorrhagic septicaemia in rabbits,which has a high morbidity and mortality rate and often leads to huge economic losses in animal husbandry.P.multocida is often mixed with other pathogens and occurs throughout the country.Establishing rapid detection method of P.multocida is a key measure for successful prevention and treatment,so this study aims to establish P.multocida TaqMan fluorescence quantitative PCR(qPCR)detection method,and carry out the epidemiological investigation of P.multocida from cattle and sheep in Ningxia.The research contents were as follows.(1)Specific primers and fluorescent labeling probes were designed according to the five capsular gene sequences(hyaC-hyaD,bcbD,dcbF,ecbJ,fcbD)of P.multocida in the NCBI database.The annealing temperature was adjusted by gradient setting,the primer and probe concentrations were optimized by matrix method,a standard curve was established for plasmids,specificity,sensitivity and repeatability tests were performed,and finally a multiplex TaqMan qPCR detection method for these five genes was established.The results showed that the sensitivity of this method was high,with the lower limit of pMD18-hyaC-hyaD detection being 5.237×102 copies/μL,the lower limit of pMD18-bcbD detection being 4.45×102 copies/μL,the lower limit of pMD18-dcbF detection being 2.89×101 copies/μL,the lower limit of pMD18-ecbJ detection being 3.74×102 copies/μL,the lower limit of pMD18-fcbD detection being 5.68×102 copies/μL,and the sensitivity was 10～100 times higher than that of ordinary PCR.The specificity was strong,and there were no amplification curves in the DNA detection of 8 pathogenic bacteria,including Bacillus subtilis,Proteus mirabilis,Staphylococcus aureus and Rhizobium radiobacter.This detection method has a good linear relationship,and the resulting linear equations were as follows:y=-3.277x+33.663,R2=0.9984,y=-3.765x+37.397,R2=0.9948,y=-3.869x+35.865,R2=0.9988,y=-3.668x+34.996,R2=0.995,y=-3.769x+35.11,R2=0.9979,the coefficient of variation of Ct value of the repeatability test between and within groups was less than 3%,indicating that the establishment of this test method has good repeatability.(2)Field epidemiological investigation was carried out on 24 large-scale farms in 8 counties and districts including Tongxin,Yanchi and Yuanzhou District in Ningxia,382 nasal swabs of cattle and sheep were collected,and the capsule serotype was analyzed through pathogen isolation and molecular biology detection,and the results showed that a total of 77 P.multocida strains were isolated from molecular biology.The total positive rate of P.multocida was 20.2%(77/382),the positive rate in each region was 12.7%～34.3%,the highest positive rate in Lingwu was 34.3%,and the lowest positive rate in Tongxin was 12.7%.The isolated strains were serotyped by the established TaqMan qPCR detection method,and the capsular serological type A 44(57%)strain,the capsular serological type B 29(38%)strain,and the capsular serological type D 4(5%)strains were obtained.The incidence of multocidal pasteurellosis in cattle and sheep was mainly concentrated in the age of 3～8 months,and the infection rate was more than 13%.In summary,this study focused on multiple TaqMan qPCR detection methods were established by five capsular genes of P.multocida,which had strong specificity and high sensitivity,and could be used for the detection and investigation of P.multocida infection;Epidemiological investigation showed that cattle and sheep in Ningxia were infected by P.multocida seriously.The results of the study has provided reference for epidemiology,source traceability,pathogen identification,and scientific prevention and control in Ningxia. |
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