2.3 Development of SYBR GreenⅠbased real-time PCR assays
To develop the SYBR Green I-based real-time PCR assays for detection of PBoV1/2 and PBoV3/4/5, the standard curves were generated. Conventional PCR amplifications with primers PBoV1/2-F/R and PBoV3/4/5-F/R were performed using the PCT-200 Peltier thermal cycler (MJ Research, USA). The positive PCR products were purified using the Gel Extraction Mini Kit (Watson Biotechnologies. INC) according to the manufacturer’s instructions, and cloned into pMD18-T vector (Takara, Dalian, China). The recombinant plasmids were designed as pMD18-327 and pMD18-209 respectively, and then transformed into Escherichia coli DH-5α competent cells (TaKaRa). Positive clones were isolated and submitted for sequencing. The concentrations and purities of the two standard plasmids were quantified by ultraviolet absorbance at 260 nm and 280 nm using a Nano-100 micro-spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The copy numbers of plasmids were determined and 10-fold serial diluted in TE buffer (10mM Tris–HCl, 1mM EDTA). The real-time PCR standard curves for detection of 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 real-time PCR reactions were optimized, and the sensitivity, specificity and reproducibility were evaluated.
The SYBR Green I-based real-time PCR amplifications of PBoV1/2 and PBoV3/4/5 were performed using the Rotor Gene 2000 real-time thermal cycler (Corbett Research, Australia). PCR was performed 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.