| Actinobacillus pleuropneumoniae,Haemophilus parasuis and Mycoplasma hyopneumoniae have similar clinical symptoms,and often form mixed infection with other respiratory pathogens,so it is very difficult to make clinical differential diagnosis.The establishment of a rapid,sensitive,high specificity and quantitative detection method is of great significance for the early diagnosis,prevention,control and purification of these three respiratory diseases.Three pairs of specific primers and three TaqMan probes are designed for ApxⅣgene of Actinobacillus pleuropneumoniae,Omp2 gene of Haemophilus parasuis and P46 gene of Mycoplasma hyopneumoniae.The TaqMan multiplex real-time quantitative PCR method is established to detect three pathogens simultaneously,and its specificity,sensitivity and repeatability are tested.The clinical samples are tested and compare with PCR.The results are as follows:TaqMan multiplex real-time quantitative PCR method is successfully established for detection of Actinobacillus pleuropneumoniae,Haemophilus parasuis and Mycoplasma hyopneumoniae There is no cross reaction between different pathogens,only Actinobacillus pleuropneumoniae,Haemophilus parasuis and Mycoplasma hyopneumoniae positive template have amplification signal.The standard curve of TaqMan multiplex qPCR method have good linear relationship,the correlation coefficient of Actinobacillus pleuropneumoniae standard curve is 0.999,Haemophilus parasuis standard curve correlation coefficient is 0.998,Mycoplasma hyopneumoniae standard curve correlation coefficient is 1.000.TaqMan multiple real-time fluorescent quantitative PCR method minimum detection copy number Actinobacillus pleuropneumoniae 9.14 x10~0 copies/μL,Haemophilus parasuis1.18 x10~1 copies/μL,Mycoplasma hyopneumoniae 1.55 x10~1 copies/μL,detection limit is 10~3times higher than conventional PCR;The coefficient of variation of the repeated test is less than 2%,and the reproducibility is good. |
