Massonet C, De Baere T, Van Eldere J
Department Medical Diagnostic Sciences, K.U. Leuven, B-3000, Leuven, Belgium.
J Microbiol Methods. 2006 Aug;66(2):369-79. doi: 10.1016/j.mimet.2006.01.001. Epub 2006 Feb 17.
We describe the development and application of a rapid and universal molecular technique for direct identification of multiple bacteria in clinical samples. Amplification of the 16S-23S rRNA spacer-region using universal primers led to fragment patterns distinct for different bacterial species and that were analyzed with fluorescent amplicon length analysis (FALA). 136 pure cultures of clinical isolates and 20 culture collection strains belonging to 22 different medically important species were used to create a primary database of fragments with sizes between 100 and 1000 bp. Subsequently, 127 respiratory samples were analyzed with culture-based techniques and via FALA of the 16S-23S rRNA spacer-region. Two DNA extraction methods were evaluated: Instagene (FALA-I) and Fastprept (FALA-P). Of the 127 samples, 26 culture-negative samples were also negative with FALA-P. Of 18 samples with growth of commensal oral flora, 10 gave a mixed oral flora pattern with FALA-P and 8 gave a negative result. For 54 samples with growth of a single bacterial species, FALA-P gave an identical result for 46. For 29 samples with growth of more than one bacterial species, identical results were obtained in 19 samples. False-negative results with FALA-P were mostly due to paucity (less than 10(3) CFU/ml) of bacteria (12 out of 18 false-negatives) or difficulties with homogenization of viscous samples (6 out of 18 false-negatives).With regard to identification of all significant pathogens of clinical samples tested, the sensitivity of FALA-P was 77% and its specificity was 100%. With FALA-I, the number of false-negative results was higher than with FALA-P due to less efficient extraction of DNA, particularly with Staphylococcal species. FALA-P allows rapid and direct identification of multiple species directly from clinical samples; pauci-cellular samples may give false-negative results.
我们描述了一种用于直接鉴定临床样本中多种细菌的快速通用分子技术的开发与应用。使用通用引物扩增16S - 23S rRNA间隔区，产生了不同细菌物种特有的片段模式，并通过荧光扩增子长度分析（FALA）进行分析。使用属于22种不同医学重要物种的136株临床分离纯培养物和20株培养保藏菌株创建了一个片段大小在100至1000 bp之间的初级数据库。随后，采用基于培养的技术以及通过对16S - 23S rRNA间隔区进行FALA分析了127份呼吸道样本。评估了两种DNA提取方法：InstaGene（FALA - I）和Fastprept（FALA - P）。在127份样本中，26份培养阴性样本用FALA - P检测也为阴性。在18份有口腔共生菌群生长的样本中，10份用FALA - P检测呈现混合口腔菌群模式，8份检测为阴性。对于54份有单一细菌物种生长的样本，FALA - P在46份样本中给出了相同结果。对于29份有不止一种细菌物种生长的样本，19份样本获得了相同结果。FALA - P的假阴性结果主要是由于细菌数量少（低于10³ CFU/ml）（18例假阴性中有12例）或粘性样本匀浆困难（18例假阴性中有6例）。关于所检测临床样本中所有重要病原体的鉴定，FALA - P的灵敏度为77%，特异性为100%。使用FALA - I时，由于DNA提取效率较低，尤其是对于葡萄球菌属物种，假阴性结果的数量高于FALA - P。FALA - P能够直接从临床样本中快速直接鉴定多种物种；细胞数量少的样本可能会给出假阴性结果。
