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首页> 外文期刊>Journal of Clinical Microbiology >Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.
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Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

机译：消除聚合酶链反应试剂中的污染DNA：对检测未培养病原体的一般方法的影响。

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摘要

Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are now feasible and may form the basis of the next generation of automated microbial identification systems. However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA. Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents. The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured.
机译：基于16S rRNA序列比较的分析提供了一种快速可靠的方法来鉴定人类病原体。通过将寡核苷酸引物指向在整个真细菌界中保守的序列，可以通过聚合酶链反应扩增几乎任何真细菌界成员的细菌16S核糖体DNA序列，然后通过序列测定进行分析。实际上，用于大范围扩增，测序和数据分析的自动化系统现在是可行的，并且可以构成下一代自动化微生物鉴定系统的基础。然而，通过该策略鉴定病原体受到用于扩增反应的试剂，特别是Taq聚合酶的频繁污染而被外源细菌DNA污染。在这里，我们描述了使用8-甲氧基补骨脂素和长波紫外光消除聚合酶链反应试剂中污染性DNA的详细研究。在未培养特异性细菌制剂的脓疱性骨髓炎的病例中，已证明了所开发方法的临床实用性。

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