Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples
Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples
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DOI：
10.1128/aem.07878-11
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发表时间：
2012-06-01
影响因子：
4.4
通讯作者：
Buergmann, Helmut
中科院分区：
文献类型：
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作者：
Brankatschk, Robert;Bodenhausen, Natacha;Buergmann, Helmut
实时定量PCR（qPCR）是微生物群落分析中广泛使用的技术，其允许定量群落样品中的靶基因的数目。目前，绝对定量的标准曲线（SC）方法被广泛用于这类分析。然而，SC方法假设标准品和样品靶模板的扩增效率（E）相同。我们在qPCR检测中分析了19种细菌菌株和9种环境样品，靶向nifH和16S rRNA基因。qPCR的E值显著不同，取决于模板。这对量化有重大影响。如果样品和标准品的E值不同，则定量误差可能高达几个数量级。为了解决这个问题，我们提出并测试了一点校准（OPC）方法的绝对定量。OPC方法校正了E的差异，并源自校正了E的Delta Delta C-T方法，该方法通常用于基因表达研究中的相对定量。SC和OPC方法进行了比较，通过定量人工模板混合物，从Gesterium sulfurreducens（DSM 12127）和念珠藻（培养物收集藻类和原生动物[CCAP] 1453/33），这在其E值不同。虽然SC方法偏离预期的nifH基因拷贝数3至5倍，但OPC方法以高准确度定量模板混合物。此外，分析环境样品，我们表明，即使很小的差异E之间的标准和样品可以导致显着差异的拷贝数计算的SC和OPC方法。
Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the Delta Delta C-T method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.