Meier A, Persing D H, Finken M, Böttger E C
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
J Clin Microbiol. 1993 Mar;31(3):646-52. doi: 10.1128/jcm.31.3.646-652.1993.
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.
基于16S rRNA序列比较的分析为鉴定人类病原体提供了一种快速且可靠的方法。通过将寡核苷酸引物指向整个真细菌界保守的序列，几乎任何真细菌界成员的细菌16S核糖体DNA序列都可以通过聚合酶链反应进行扩增，随后通过序列测定进行分析。实际上，用于广泛扩增、测序和数据分析的自动化系统现在是可行的，并且可能构成下一代自动化微生物鉴定系统的基础。然而，这种策略在鉴定病原体时受到用于扩增反应的试剂（特别是Taq聚合酶）频繁被外源细菌DNA污染的阻碍。在此，我们描述了关于使用8-甲氧基补骨脂素和长波紫外线消除聚合酶链反应试剂中污染DNA的详细研究。在一例少菌型骨髓炎病例中证明了所开发程序的临床实用性，该病例未培养出特定的细菌病原体。
