Establishment And Application Of 5-plex PCR Method For Detection Of Porcine Respiratory Pathogenic Bacteria

Pasteurella,Streptococcus suis,Mycoplasma hyopneumoniae,Actinobacillus pleuropneumoniae and Haemophilus parasuis are the pathogens of swine pasteurellosis,streptococcal disease,mycoplasmal pneumonia of swine,porcine contagious pleuropneumonia and Glasser's disease,respectively.They are also the most common bacterial pathogens in the pig industry.These pathogens cause respiratory diseases of pigs.The clinical symptoms and pathological changes cannot be distinguished by clinical observation.Mixed infections gives more obstacles for the clinical diagnosis.In order to effectively identify these pathogens,this subject intends to establish a PCR diagnostic method to identify these five pathogens simultaneously.1.Establishment of single-plex PCR detection method for porcine respiratory pathogenic bacteriaObjective: To establish single PCR methods for detection of porcine respiratory pathogens.Methods: Five pairs of specific primers for the plpE gene of Pasteurella,the gdh gene of S.suis,the mhp677 gene of M.hyopneumoniae,the apx ? gene of A.pleuropneumoniae,and the vanY gene sequence of H.parasuis were designed.Optimal annealing temperatures were determined for each PCR.Meanwhile,the sensitivity and the specificity of primers were also tested.Results: The annealing temperature was ranged between 54? and 60?.The primers of each gene were specific to each pathogen.The minimum detection limits of plpE gene was 0.01 ng/?L,gdh gene was 0.1 ng/?L,mhp677 gene was 0.1 ng/?L,apx? gene is 0.1 ng/?L,and vanY gene was 1 ng/?L.Conclusion: The single PCR detection methods for the five pathogens were established,which laid the foundation for the establishment of the 5-plex PCR detection methods.2.Establishment of single PCR detection method for porcine respiratory pathogenic bacteriaObjective: To establish a 5-plex PCR detection method for detection of porcine respiratory pathogens.Methods: All primers determined in single PCRs and templates were mixed to develop 5-plex PCR method.Optimal annealing temperatures were determined for 5-plex PCR.Meanwhile,the sensitivity and the specificity of primers were also tested.Results: The annealing temperature was determined as 60?.The primers of each gene were specific to each pathogen.The minimum detection limits of plpE gene was 1 ng/?L,gdh gene was 10 ng/?L,mhp677 gene was 1 ng/?L,apx? gene is 10 ng/?L,and vanY gene was 10 ng/?L.Conclusion: A 5-plex PCR method for the detection of 5 swine respiratory pathogens was established.3.Application of 5-plex PCR detection method for porcine respiratory pathogenic bacteriaObjective: To verify whether the established 5-plex PCR method can be used for clinical detection.Methods: Pathological lung tissues were collected and the genome of each sample was extracted.Five pathogens,including Pasteurella,S.suis,M.hyopneumoniae,A.pleuropneumoniae and H.parasuis were detected by the established 5-plex PCR method.Results: Two hundred and twenty four lung tissue samples were collected.Sixty five samples were proved to be positive,including 50 single infectious samples and 15 multiple infectious samples.Conclusion: The 5-plex PCR that we established could be used for the detection and pathogenic epidemiological survey of Pasteurella,S.suis,M.hyopneumoniae,A.pleuropneumoniae and H.parasuis.