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

For the purpose of validation, real-time PCR reliability, sensitivity, and linearity of standard curves were verified by testing tenfold serial dilutions of synthesized DNA, including each target genome sequence (1 × 10 0 to 1 × 10 6 copies/reaction). The synthesized DNA was purchased from Integrated DNA Technologies (Integrated DNA Technologies, Inc.). Pathogen dilutions were repeated twice in separate runs, and a standard curve was constructed from the Cq values. The PCR efficiency (E) was calculated using the standard curve slope according to the following formula: E=(10 −1/slope (−1) ). The correlation co-efficient (R 2 ) was also calculated. The limit of detection (LOD) was defined as the lowest concentration at which a fluorescent signal could be detected in all reactions. Reproducibility (inter-assay variance) was assessed using the coefficient value (CV) calculated based on quantification cycle (Cq) values.