期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 98, 期 13, 页码 6105-6113出版社
SPRINGER
DOI: 10.1007/s00253-014-5794-4
关键词
Droplet digital PCR; Quantitative real-time PCR; Population dynamics; Soil microorganism
资金
- National Research Foundation of Korea (NRF) grant - Korean government (MSIP) [2012R1A2A03046724]
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 (R (2) = 1.00) and PCR efficiency (a parts per thousand yen92 %) 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 similar to 5 x 10(8) and 0 similar to 5 x 10(7) 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 mu L DNA extract(-1) as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies mu L DNA(-1)) at a parts per thousand yen5 x 10(5) 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 center dot x) revealed excellent quantitative agreement between the two technologies (a = 0.98, R (2) = 0.97 in the CupMBT set and a = 0.90, R (2) = 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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