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

A total of 17 primer-probe sets were used to detect pathogens that certainly or possibly cause respiratory diseases on porcine. Each primer-probe set was designed to detect a single target pathogen. New primer-probe sets were designed for Pasteurella multocida and toxin, M. hyosynovie, PCV2, PCMV, SIV and PHEV using the PrimerQuest software (Integrated DNA Technologies, Inc., Coralville, IA, USA) based on consensus sequences of each pathogen obtained from the GenBank database. Primer and probe information and their target pathogens are summarized in Table 1 . GenBank accession numbers, the reference sequence, country, host and first deposited year used for primers and probes design of each pathogen were shown in Supplemental Table 1 . Previously reported qPCR assays were used for 10 pathogen species, including RNA, DNA viruses and bacteria [1, 2, 12, 16, 18, 19, 25, 27, 28] . Furthermore, as an internal control within the Dempo-PCR reaction, primer-probe sets for β-actin were synthesized as previously reported [29] . All hydrolysis probes were labeled with the reporter dye FAM (6-carboxyfluorecein) at the 5′ end and the fluorescent dye TAMRA (6-carboxytetramethylrhodamine) at the 3′ end. Primers and probes were purchased from Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA), and probes containing the mixed base were produced at Integrated DNA Technologies (Integrated DNA Technologies, Inc.).