Establishment And Application Of Multiplex PCR Detection Method For Porcine Respiratory Pathogens

In order to rapidly diagnose porcine respiratory diseases caused by Streptococcus suis（SS）,Actinobacillus pleuropneumoniae（APP）,Pasteurella multocida（Pm）,Haemophilus parasuis（HPS）,Bordetella bronchiseptica（Bb）and Mycoplasma pneumoniae（Mhp）,and to investigate the prevalence of pathogens in the respiratory tract of pigs in Guizhou Province.The specific primers of APP ApxⅣA gene,Pm KMT1 gene,SS gdh gene,HPS 16S r RNA gene and Mhp P36 ldh gene were designed and synthesized respectively,and the primer of Bb fla gene was synthesized by referring sequences in references.Optimizing single and multiplex PCR reaction system and reaction conditions,the single and multiplex PCR detection methods were established,and the specificity,sensitivity and reproducibility of methods were tested.And 25 nasal swabs of diseased pigs,14 lung tissue samples of diseased pigs and 430nasal swabs of apparently healthy pigs,which were collected from several pig farms of 9 cities（prefectures）in Guizhou Province from October 2020 to March 2021,were detected by the established six-plex PCR and single PCR methods.The results showed that:1.A single PCR detection methods for APP,Pm,SS,HPS,Bb and Mhp were successfully established,which can be used to quickly diagnose bacterial respiratory diseases in pigs.The optimal concentration of upstream and downstream primers for APP,Pm,SS,HPS,Bb and Mhp was 0.6μmo L·L^-1、0.4μmo L·L^-1、0.4μmo L·L^-1、0.4μmo L·L^-1、0.6μmo L·L^-1、0.4μmo L·L^-1respectively,the optimal annealing temperature was 62℃,and the minimum amount of nucleic acid detected by single PCR was APP 0.07 pg,Pm 0.044 pg,SS 3.06 pg,HPS 1.90 pg,Bb 4.44 pg,Mhp 6.14pg;and the specificity was quite well,only the specific target gene fragments of APP,Pm,SS,HPS,Bb and Mhp were amplified.2.Two-plex PCR for APP and HPS,three-plex PCR for APP,SS and HPS,four-plex PCR for APP,SS,HPS and Bb,five-plex PCR for APP,Pm,SS,HPS and Bb,and the specificity,sensitivity and reproducibility of these multiplex PCR detection methods were quite well.Six-plex PCR for APP,Pm,SS,HPS,Bb and Mhp were successfully established,the optimal concentration of upstream and downstream primers of six-plex PCR were that:apxⅣA-F/R 1.0μmo L·L^-1,KMT1-F/R 0.2μmo L·L^-1、gdh-F/R 0.2μmo L·L^-1、16S r RNA-F/R 0.2μmo L·L^-1、p36 ldh-F/R 0.2μmo L·L^-1and fla-F/R 0.4μmo L·L^-1,the optimal annealing temperature was 62℃,and the minimum amount of nucleic acid detected simultaneously by six-plex PCR was:APP 26.25 pg,Pm 2.19 pg,SS 2.0 pg,HPS 2.22 pg,Mhp 3.07 pg,Bb 4.3 pg.Moreover,the specificity and reproducibility of these multiplex PCR detection methods were quite well.When the six-plex PCR established in this study was used in the simultaneous detection of clinical samples,the coincidence rate with the number of positives detected by the single PCR detection method is 96.76%.3.October 2020 to March 2021,the prevalence of porcine respiratory pathogens of several pig farms in Guizhou Province was dominated by SS and HPS,and the type of infection was mainly SS+HPS double infection,other infection types with higher detection rates were SS,Pm+SS+HPS and HPS,five pathogens detected in the same sample were only detected from apparently healthy pig nose swabs,and APP was mainly detected from the lungs of diseased pigs.The respiratory pathogen infection types of the samples,collected from some pig farms in 9 cities（prefectures）from October 2020 to March 2021,was the most in Liupanshui City,followed by Tongren City and Qiandongnan Prefecture,and it was the least in Guiyang city.In addition to Zunyi City,Qianxinan Prefecture and Qiandongnan Prefecture,the respiratory pathogen infection type of some pig farms in the other six cities（prefectures）was mainly SS+HPS,and the respiratory pathogen infection type of some pig farms was mainly SS in Qiandongnan Prefecture.