Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil
Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil
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DOI：
10.1007/s00253-014-5794-4
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发表时间：
2014-07-01
影响因子：
5
通讯作者：
Cho, Kyung-Suk
中科院分区：
文献类型：
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作者：
Kim, Tae Gwan;Jeong, So-Yeon;Cho, Kyung-Suk
微滴数字PCR（DD-PCR）技术作为一种DNA定量技术在医学诊断领域有着广阔的应用前景。本研究以实时荧光定量PCR（qRT-PCR）为参考技术，评价了DD-PCR作为土壤DNA定量工具的适用性。使用贪铜菌属MBT 14和鞘氨醇菌属MD 2，并为每种设计引物/TaqMan探针组（分别为CupMBT和SphMD 2）。对10倍稀释系列的标准曲线分析表明，qRT-PCR和DD-PCR在其检测范围内均表现出良好的线性（R（2）= 1.00）和PCR效率（千分之92%）。然而，DD-PCR显示出比qRT-PCR高10倍的灵敏度。将MBT 14和MD 2分别以每克土壤约5 × 10（8）和约5 × 10（7）个细胞的浓度添加到非无菌土壤中（n = 5）。该细菌负荷试验表明，DD-PCR比qRT-PCR更敏感和更具区分性。例如，DD-PCR显示随着细菌负荷的增加，DNA从14逐渐增加到141，160 MBT 14 rDNA拷贝μ L DNA提取物（-1），而qRT-PCR可以定量DNA（6，432拷贝μ L DNA（-1）），单位为每千日元5 × 10（5）MBT 14/克土壤。当在改良土壤中监测3周的时间DNA变化时，两种技术随着时间的推移表现出几乎相同的变化。线性测试（y =中心点x）显示两种技术之间具有极好的定量一致性（CupMBT组中a = 0.98，R（2）= 0.97，SphMD 2组中a = 0.90，R（2）= 0.94）。这些结果表明，DD-PCR是一种很有前途的工具，可以检测复杂环境中微生物的时间动态。
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.