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首页> 外文期刊>Journal of Clinical Microbiology >Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate
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Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate

机译：去除PCR抑制剂聚腺苷磺酸钠可改善血液培养物中微生物DNA的扩增

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Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted E. coli 16S rRNA gene was achieved by adding 5 μl of undiluted processed sample DNA to a 50-μl PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.
机译：分子方法越来越多地用于鉴定临床样品中的微生物。 PCR的常见技术问题是由于PCR抑制剂的存在导致扩增失败。由于该原因，从接种的血液培养基中扩增细菌16S rRNA基因的最初尝试失败。尽管进行了许多纯化DNA的尝试，但该抑制剂仍然存在，并且被确定为聚蒽硫磺酸钠（SPS），这是血液培养基的常见添加剂。像DNA一样，SPS是一种高分子量聚阴离子，可溶于水，但不溶于醇。因此，SPS倾向于与DNA共纯化。设计了一种提取方法，用于从血液培养基中纯化DNA并去除SPS。用苄醇对含有人血并掺有大肠杆菌的血液培养基进行有机萃取，并用分光光度法记录了SPS的去除。成功扩增提取的 E。大肠杆菌16S rRNA基因是通过将50 µl PCR混合物中加入5 µl未稀释的处理过的样品DNA来获得的。当使用其他纯化方法时，仅通过稀释这些样品即可克服SPS的抑制作用。通过我们的提取技术，甚至在未经接种的血液培养基中进行大范围16S rRNA基因共有PCR时，也含有细菌DNA。我们得出的结论是，血液培养添加剂SPS是PCR的有效抑制剂，可以抵抗传统DNA纯化方法的去除，但可以通过苯甲醇提取方案去除，从而提高了PCR性能。

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