Quantitative PCR assays

TaqMan based quantitative PCR assay for V. parahaemolyticus and SYBR green based qPCR assays for HBx and presumptive protist were conducted (Table 1) . All qPCR assays were performed using the BioRad Real-Time PCR system. For TaqMan based qPCR, the reaction mixture (20 µl) contained 1x PerfeCTa qCPR ToughMix (Quanta Biosciences, Beverly, MA), 0.5 µM of each primer, and 2.5 µl of the template DNA. QPCR reactions were performed in duplicate and amplification protocols consisted with a hold at 95 °C for 2 min, followed by 45 cycles of 94 °C 5s, 59 °C 45 s. SYBR green-based qPCR reaction mixture (15 µl) contained 1x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA), 0.5 µM of each primer, and 2.5 µl of the template DNA. The amplification conditions for HBx consisted of a hold at 94°C for 2 min, followed by 45 cycles of 94°C 30s, 62°C 10 s, and 72°C 10 s. The amplification conditions for presumptive protist consisted of a hold at 94°C for 2 min, followed by 30 cycles of 94°C 30s, 55°C 30 s, and 72°C 45 s.

A calibration curve with concentrations spanning the range from 10 to 106 gene copies per reaction, with two replicates, was prepared. Duplicate no-template controls (NTC) were included in each run. The amplification efficiencies (AE) were calculated based on the equation: AE= 10(-1/slope) –1. A summary of qPCR target organisms, primer/ probe name, and sequences are detailed in Table 1.

About the lower limit of detection (LLOD): Theoretically the LLOD for qPCR is 3 gene copies per reaction. In our experiment, we use the lowest point of our standard curve as the lower limit of quantification (LLOQ) which is 10 gene copies per reaction. The LLOD can also be calculated based on the methods mentioned in these two papers: Staley et al. (2012) and Xue et al (2019): which is the inverse log of five times the difference between the Y-intercept and the upper 99% confidence interval of the intercept divided by the slope based on the standard curve.

The LLOD for flow cytometry is 2 cells per ml.

protist_predation.csv