Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs

PIEWBANG, Chutchai; RUNGSIPIPAT, Anudep; POOVORAWAN, Yong; TECHANGAMSUWAN, Somporn

Optimization of the simplex PCR: Prior to performing the PCR for detection of RNA viruses, a first round PCR for CRCoV was performed in order to increase the detection sensitivity. Reactions were comprised of a mixture of 2x GoTaq ® Hot Start Green Master Mix (Promega, Madison, WI, U.S.A.), 0.4 µM final concentration of each outer primer (CoV_16053_F and CoV_16594_R) and 2 µl of cDNA, and made up to 25 µl with nuclease-free water. Reactions were performed using 3Prime G Gradient Thermal Cycle (Techne, Bristol, U.K.). Cycling conditions were comprised of an initial denaturation at 94°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec. The final extension was performed at 72°C for 7 min. Subsequently, the amplified CRCoV product of the first round PCR, and cDNA of the other RNA viruses (CIV, CPIV and CDV) and extracted DNA viruses (CAdV-2 and CaHV-1) were used as a template for further simplex PCR studies. Gradient simplex PCR was performed for each virus. All reaction compositions were as mentioned above, but the gradient annealing temperature (Ta) was programmed ranging from 50°C to 59°C in order to optimize the reaction. Thermal cycling was performed with 95°C for 5 min, then 40 cycles of 95°C for 1 min, varied Ta for 1 min and 72°C for 1 min, and then finally 72°C for 10 min. The amplicons were resolved by 2% (w/v) agarose gel electrophoresis with 10% ethidium bromide in-gel staining and visualized by UV transillumination and compared to expected size of the PCR product ( Table 1) .