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

To evaluate the sensitivity, linearity, and efficiency of the PCR, 10-fold serial dilutions of synthesized DNA were tested by realtime PCR. Standard curves were constructed from Cq values, then the LOD, R 2 , and E were evaluated ( Supplementary Fig. 1 ). Table 2 shows the results for LOD number and CVs of run-to-run variants. The LOD, based on DNA copy number, was ≤100 copies/reaction for all primer-probe sets. The CVs were at most 2.62%; this reproducibility was observed with PRCV testing. In addition, the calibration curves of all assays covered a linear dynamic range of more than five orders of magnitude and showed R 2 values of at least 0.9922. Although the PCR efficiency for PRRSV US strain and PRV was slightly low (81.6% and 88.6%, respectively), the PCR efficiency in all detection assays was more than 80%, which was enough to quantify the target copy number.