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

Step PrimeScript RT-PCR Kit (Perfect Real Time) (TaKaRa Bio, Kusatsu, Japan) was used to detect viral RNA, and Premix Ex Taq (Perfect Real Time) (TaKaRa Bio) was used to detect viral and bacterial DNA. All reactions were performed in a total volume of 20 µl, which contained the sample nucleic acid, primers, probes (the final concentration of all primers and probes was 0.2 µM) and all other components included in the kits, according to the manufactures' protocols. Thermal cycling conditions were as follows: 45°C for 5 min and 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec, 55°C for 20 sec, and 72°C for 20 sec [26] . Fluorescent signal data were analyzed using an automatic quantification algorithm in LightCycler Nano Software 1.1 (Roche Diagnostics GmbH), and the parameters of analysis were as follows: exclude early cycle=7, minimum relative amplifications=0, and minimum amplification quality=5.