| The purpose of this study was to establish a quintuple PCR method for rapid detection of porcine respiratory pathogenic bacteria, including Pasteurella, Streptococcus suis, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae and Glaesserella parasuis. Five pairs of specific primers were designed according to the sequences of plpE gene of Pasteurella, gdh gene of S. suis, mhp677 gene of M. hyopneumoniae, apxⅣ gene of A. pleuropneumoniae, and vanY gene of G. parasuis published in GenBank database. A quintuple PCR method was developed to detect the above 5 pathogenic bacteria simultaneously. The amplification condition, specificity and sensitivity were optimized. Moreover, pathogens were detected by using the established quintuple PCR method from 224 lung tissue samples with pathological changes. The results showed that the established quintuple PCR method had good specificity and certain sensitivity, and could amplify the specific fragments of plpE (1 001 bp), gdh (689 bp), mhp677 (526 bp), apxⅣ (417 bp), and vanY (302 bp) simultaneously. Meanwhile, the quintuple PCR method was negative for the amplification of other porcine pathogens. The minimum detection limits of Pasteurella, S. suis, M. hyopneumoniae, A. pleuropneumoniae and G. parasuis were 3.72×105 copies/μL, 4.18×105 copies/μL, 1.01×106 copies/μL, 3.92×106 copies/μL, and 3.98×106 copies/μL, respectively. At least one pathogen was detected in 65 of the 224 lung samples (29.0%), including 50 samples (22.3%) infected by one pathogen and 15 samples (6.7%) infected by more pathogens. These results proved that the quintuple PCR method established in this study can be used to detect 5 pathogenic bacteria in clinical lung samples. |
