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

The assay was applied to test clinical samples. A total of 30 samples of porcine lung tissue submitted in 2016-2018 to Azabu University for diagnosis of porcine respiratory pathogens were used to test. These pigs were 48 to 135 days old and belonged to 6 farms (A to F), all showing respiratory symptoms (Supplementary Table 3 ). Lung tissues were minced by scissors, diluted 1:10 in phosphate buffered saline (PBS, pH 7.4), homogenized for 20 sec at 3,200 rpm with the presence of three stainless steel beads (φ4 mm) by using the bead crusher µT-12 (TAITEC, Inc.), and centrifuged at 12,000 g for 5 min to obtain the supernatant. Bacteria nucleic acids, viral DNA, and viral RNA were extracted from the supernatant using a QIAamp ® cador ® Pathogen Kit (Qiagen, Hilden, Germany) with a sample volume of 200 µl and elution volume of 50 µl, as described by the manufacturer. The extracted DNA and RNA were stored at −80°C until examination. The extracted nucleic acids were evaluated in triplicated by targeting respiratory disease complex pathogens in a single run of Dempo-PCR. When the Cq values were calculated by algorithm described above in more than two out of three runs, the samples were considered positive. In order to compare Dempo-PCR assay with the classical method, the conventional PCR (cPCR) was performed under each condition using conventional primers (Supplementary Table 2 ). A PrimeScript TM RT Master Mix (TaKaRa Bio) and GoTaq ® Green Master Mix (Promega) was used. All reactions were performed in a total volume of 25 µl, which contained the sample nucleic acid, primers (the final concentration of all primers was 0.4 µM) and all other components included in the kits, according to the manufactures' protocols. Amplicons were detected by electrophoresing. The samples which showed the results of the Dempo-PCR assay is not consistent with the cPCR assay did not match, were confirmed by direct sequencing of amplification products.