比较使用实时荧光定量 PCR 检测组织中布鲁氏菌的商用 DNA 提取试剂盒。
Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR.
机构信息
Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburgerstrasse 96a, 07743 Jena, Germany.
出版信息
BMC Infect Dis. 2010 Apr 20;10:100. doi: 10.1186/1471-2334-10-100.
BACKGROUND
The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA.
METHODS
Five kits were evaluated using clinical specimens: QIAamp DNA Mini Kit (QIAGEN), peqGold Tissue DNA Mini Kit (PeqLab), UltraClean Tissue and Cells DNA Isolation Kit (MoBio), DNA Isolation Kit for Cells and Tissues (Roche), and NucleoSpin Tissue (Macherey-Nagel). DNA yield was determined using a quantitative real-time PCR assay targeting IS711 that included an internal amplification control.
RESULTS
Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p < 0.05) and of diagnostic relevance. Sample volume, elution volume, and processing time were also compared.
CONCLUSIONS
We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.
背景
由于细菌数量通常较低，使用 PCR 检测组织标本中的布鲁氏菌较为困难。因此，优化的 DNA 提取方法至关重要。本研究旨在评估商业试剂盒提取布鲁氏菌 DNA 的性能。
方法
使用临床标本评估了五种试剂盒：QIAGEN 的 QIAamp DNA Mini Kit、PeqLab 的 peqGold Tissue DNA Mini Kit、MoBio 的 UltraClean Tissue 和 Cells DNA Isolation Kit、Roche 的 DNA Isolation Kit for Cells and Tissues，以及 Macherey-Nagel 的 NucleoSpin Tissue。使用包含内部扩增对照的针对 IS711 的定量实时 PCR 检测法确定 DNA 产量。
结果
QIAGEN 和 Roche 的试剂盒提供的 DNA 量最高，Macherey-Nagel 和 Peqlab 的产品处于中间水平，而 MoBio 的试剂盒产生的 DNA 量最少。差异具有统计学意义（p < 0.05）且具有诊断意义。还比较了样品体积、洗脱体积和处理时间。
结论
对于一些样本，最好和最差的 DNA 提取试剂盒之间的 DNA 产量差异高达两个数量级，并且偶尔会观察到抑制现象。这表明，当组织中的 DNA 含量非常低时，DNA 纯化可能比预期的更为重要。
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