Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases

SUNAGA, Fujiko; TSUCHIAKA, Shinobu; KISHIMOTO, Mai; AOKI, Hiroshi; KAKINOKI, Mari; KURE, Katsumasa; OKUMURA, Hanako; OKUMURA, Maho; OKUMURA, Atsushi; NAGAI, Makoto; OMATSU, Tsutomu; MIZUTANI, Tetsuya

In this study, Dempo-PCR has been developed, following the methods of diagnosis of bovine diarrhea developed by Tsuchiaka et al. [26] . Since all primer-probe sets were optimized in the same temperature conditions, Dempo-PCR can detect a total of 17 pathogens, including 8 viruses, 8 bacteria, and 1 toxin, in a single run of TaqMan real-time PCR. In sensitivity test by using standard curves from synthesized DNA, all primer-probe sets showed high sensitivity. Furthermore, the results of detection of target pathogens from clinical samples using this method showed similar results to the respective conventional PCR method. However, PRRS virus is known to have a high frequency of genetic mutations, and the primer and probe sequences will need to be checked at a considerable frequency when performing Dempo-PCR from field samples. The type of pathogens involved in PRDC is specific to the regions and countries where production occurs [20] . Therefore, it may be necessary to change the inspect pathogens according to the regions. However, Dempo-PCR is possible to detect many types of pathogens simultaneously.