The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R2=1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0∼5×10 8 and 0∼5×107 cells per gram of soil, respectively (n=5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies μL DNA extract -1 as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies μL DNA-1) at ≥5×105 MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y=a·x) revealed excellent quantitative agreement between the two technologies (a=0.98, R2=0.97 in the CupMBT set and a=0.90, R2=0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.
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Acknowledgments This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (No. 2012R1A2A03046724).
- Droplet digital PCR
- Population dynamics
- Quantitative real-time PCR
- Soil microorganism