Routes to improving the reliability of low level DNA analysis using real-time PCR.
Routes to improving the reliability of low level DNA analysis using real-time PCR.
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使用实时PCR提高低水平DNA分析可靠性的途径。
DOI：
10.1186/1472-6750-6-33
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发表时间：
2006-07-06
影响因子：
3.5
通讯作者：
Keer JT
中科院分区：
文献类型：
--
作者：
Ellison SL;English CA;Burns MJ;Keer JT
利用实时定量聚合酶链式反应在低水平准确定量DNA对于临床、环境和法医应用越来越重要。在低浓度水平(这里指的是低于100个目标拷贝)，DNA定量对制备过程中的损失很敏感，并且由于采样原因而存在明显的有效漏检率。本文报道了一种针对人SRY基因某一区域的实时定量聚合酶链式反应的研究，该方法的浓度范围为0.5到1000个拷贝。考察了不同的样品制备和校正方法对定量准确度的影响。在非常低的目标浓度0.5-10基因组当量(G.E.)消除每个DNA标准浓度内的任何重复，而没有可测量的信号(非检测)会影响校准。改进的校准可以通过消除任何校准标准浓度的所有校准重复而不被检测到(“通过样品消除”)来实现。如果在平均之前去除未检测到的样本，则测试样本也显示正偏差；通过转换为浓度，包括未检测到的为零浓度，并对所有值进行平均，可获得较少偏差的结果。管状塑料在低浓度下对DNA定量有极显著的影响(p=1.8×10-4)。在低浓度下(低于10G.E.)，与低保留率塑料相比，用标准塑料制备的分析结果减少了约50%。在本研究中，未发现制剂溶液(载体DNA或稳定剂)有显著影响。检测概率用Logistic回归计算。在大浓度范围内的Logistic回归被证明对由于高浓度下扩增失败而未检测到的复制反应敏感；可以通过与对数(浓度)的回归或更好地通过消除无效反应来降低效果。建议使用低保留率的塑料管对低于100G.E.的DNA溶液进行定量。对于使用线性最小二乘的低水平校准，对于显示合理地归因于采样效应的未检测的任何标准，最好消除整个重复组，而不是消除未检测或指定任意的高Ct值。在计算有无检出物的低水平测试样品的浓度时，应计算每次重复的浓度，分配给无检出物的零浓度，并取所有产生的浓度值的平均值。Logistic回归是估计低DNA浓度下检测概率的一种有用方法。
Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated. At very low target concentrations of 0.5–10 genome equivalents (g.e.) eliminating any replicates within each DNA standard concentration with no measurable signal (non-detects) compromised calibration. Improved calibration could be achieved by eliminating all calibration replicates for any calibration standard concentration with non-detects ('elimination by sample'). Test samples also showed positive bias if non-detects were removed prior to averaging; less biased results were obtained by converting to concentration, including non-detects as zero concentration, and averaging all values. Tube plastic proved to have a strongly significant effect on DNA quantitation at low levels (p = 1.8 × 10-4). At low concentrations (under 10 g.e.), results for assays prepared in standard plastic were reduced by about 50% compared to the low-retention plastic. Preparation solution (carrier DNA or stabiliser) was not found to have a significant effect in this study. Detection probabilities were calculated using logistic regression. Logistic regression over large concentration ranges proved sensitive to non-detected replicate reactions due to amplification failure at high concentrations; the effect could be reduced by regression against log (concentration) or, better, by eliminating invalid responses. Use of low-retention plastic tubes is advised for quantification of DNA solutions at levels below 100 g.e. For low-level calibration using linear least squares, it is better to eliminate the entire replicate group for any standard that shows non-detects reasonably attributable to sampling effects than to either eliminate non-detects or to assign arbitrary high Ct values. In calculating concentrations for low-level test samples with non-detects, concentrations should be calculated for each replicate, zero concentration assigned to non-detects, and all resulting concentration values averaged. Logistic regression is a useful method of estimating detection probability at low DNA concentrations.
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影响因子：
3.4
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