Taq master mix

We carried out PCR with genomic DNA from the giant panda “Panpan” to evaluate the sensitivity and specificity of the primers, as well as the optimum annealing temperatures. The microsatellite primers used in this study are listed in Table 2. The details of the PCR process are as follows: 94 °C for 3 min, followed by 35 cycles at 94 °C for 30 s, 30 s at the annealing temperature, 72 °C for 30 s, and 72 °C for 10 min in the final extension step. Each PCR reaction mixture contained 5 μL 2× Taq master Mix (Shanghai Generay Biotech, Shanghai, China), 0.4 μL of each primer (10 μM), and 0.5 μg of genomic DNA in a total volume of 10 μL. PCR products were visualized on a 1% agarose gel.
Subsequently, 21 pairs of primers with high sensitivity and specificity were used for microsatellite polymorphism analysis. The fluorescence-labelled forward primers (tetrachloro-6-car-boxyfluorescein (TET), 6-carboxyfluorescein (FAM), and hexachloro-6-car-boxyfluorescein (HEX) dyes) were synthesized and used for the PCR with genomic DNA from 20 giant pandas. PCR products were diluted 1:10 and run using the 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA) with the GeneScan 500 ROX Size standard (Applied Biosystems). The output data were analyzed using Gene Mapper 4.1 (Applied Biosystems) to assign the genotype to each sample at each locus.

Triplex PCR was performed to detect the toxR gene specific to the Vibrio species, the serogroup O3-specific gene vp0209 and the serovar K6-specific gene vp0230 according to Zhang’s protocol [22 (link)-24 ]. The forward (F) and reverse (R) primers of the three detected genes are listed in Table 1. These oligonucleotide primers were synthesized by Sangon Biotech (China). The amplification conditions used for the triplex PCR were set up according to the protocol of Zhang et al. with slight modifications [24 ]. In brief, the 25 μl reaction mixture was composed of 12.5 μl of 2× Taq Master Mix (Shanghai Generay Biotech Co., Ltd.), each primer at 0.5 μM, 6.5 μL of ddH₂O, and 2 μl of DNA template. The thermal cycling parameters for PCR were as follows: denaturation at 95°C for 2 min, 30 cycles of 95°C for 30 sec, an annealing temperature of 56°C for 30 sec, and 72°C for 1 min, with a final extension of 72°C for 5 min. PCR was performed on a Bio-Rad PTC-200 Thermal Cycler (Bio-Rad, USA). The amplified products were analyzed electrophoretically on a 1.2% agarose gel containing Gel Stain (Sangon Biotech, China) and photographed using an Amersham Imager 600 UV (GE Healthcare, USA).