Nuclease and protease free water

The DNA of meats (beef, mutton, pork, chicken and duck) was diluted to 20 ng/μL, 10 ng/μL, 5 ng/μL, 2 ng/μL, 1 ng/μL, 0.5 ng/μL, 0.1 ng/μL, and 0.01 ng/μL in turn, and the sensitivity and limit of detection (LOD) were assayed using five pairs of primers.
The 20 μL ddPCR mixture consisted of 1.8 μL forward and reverse primers (final concentration, 900 nM), 0.5 μL of the probe (final concentration, 250 nM), 10 μL of ddPCR Master Mix (Bio-Rad, Hercules, CA, USA), 1 μL of template DNA, and 4.9 μL of nuclease- and protease-free water (Thermo Scientific).
After the PCR, the droplet reading and analysis system can automatically read fluorescence signals in the droplet number, then calculate the copy number in the PCR system according to the Poisson distribution principle and divide the copy number by the volume of the PCR system to obtain the lowest copy number that can be detected by the ddPCR.

Each 20 μL reaction mixture was prepared as follows: 1.8 μL of each primer (final concentration, 900 nM), 0.5 μL probe (final concentration, 250 nM), and 10 μL ddPCR Master Mix (Bio-Rad, Hercules, CA, USA) were mixed, and then 4 μL (40-fold diluted from the original DNA extraction sample) of template DNA and 1.9 μL of nuclease- and protease-free water (ThermoScientific, Salt Lake City, UT, USA) were added. A Bio-Rad QX100 ddPCR droplet generator (Bio-Rad) was used to divide the 20 μL mixture into approximately 20000 droplets, with the target DNA segments and PCR reagents being randomly distributed among the droplets. Conventional PCR was performed using a T100 Thermal Cycler (Bio-Rad) according to the following cycling protocol: enzyme activation for 10 min at 95°C, followed by 40 cycles of 30 sec denaturation at 94°C; 1 min annealing and extension at 60°C, followed by enzyme inactivation at 98°C for 10 min and hold at 4°C (according to the manufacturer's instructions). After PCR amplification, the droplet reader determines which droplets contain the target DNA amplicon and which do not. The software then calculates the concentration of the target DNA in copies per microliter from the fraction of positive reactions using Poisson distribution analysis.

Real-time PCR was performed with in a 25 μl reaction volume, consisting of 1 μl of each primer (10 μmol/L), 0.5 μl probe (10 μmol/L), 12.5 μl real-time PCR Master Mix (Bio-Rad, Hercules, CA, USA), 5 μl nuclease- and protease-free water (Thermo Scientific, Salt Lake City, UT, USA), and 5 μl of sample DNA or plasmid DNA. For amplification, different real-time PCR instruments and software versions (ABI7300 v 1.3.1, ABI7500 v 2.3, ABI ViiA7 v 1.2, ABI7900HT v 2.3, Bio-Rad CFX96 v 3.1 and Roche Light Cycler 480 v 1.5) were used in the collaborative trial. The thermal cycling program included an initial denaturation step at 95°C for 10 min, followed by 45 cycles of 15 s at 95°C for denaturation, and 60 s at 60°C for annealing and extension. The fluorescent signal was collected after the extension step in each cycle.