2.3 SYBR Green I-based quantitative PCR assays
The quantitative PCR standard curves of detecting PBoV1/2 and PBoV3/4/5 were generated with a known copy number (from 1×109 to 1×100 copies/μL) of the recombinant plasmids pMD18-327 and pMD18-209, and performed in triplicate, respectively. The parameters of the SYBR Green I-based quantitative PCR (qPCR) reactions were optimized, and the sensitivity, specificity and reproducibility were evaluated.
The qPCR was performed using the Rotor Gene 2000 real-time thermal cycler (Corbett Research, Australia). qPCR protocol was executed in a 20-μL volume containing 3 μL DNA of samples, 10 μL of 2×SYBR Premix ExTaq I, 0.5 μL (25 μmol/L) of each primer and 6 μL distilled water. The amplification conditions were 95°C for 3 min as initial denaturation followed by 39 cycles of 95°C for 30 s, 58°C for 25 s and 72°C for 30 s.