Apfalter Petra, Barousch Wolfgang, Nehr Marion, Makristathis Athanasios, Willinger Birgit, Rotter Manfred, Hirschl Alexander M
Department of Hygiene and Medical Microbiology, University of Vienna, Vienna, Austria.
J Clin Microbiol. 2003 Feb;41(2):592-600. doi: 10.1128/JCM.41.2.592-600.2003.
Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10(-6) inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompA-based real-time protocol produced the most consistent positive results for all replicates tested down to 10(-6) IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n = 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10(8) particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n = 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.
肺炎衣原体是一种重要的呼吸道病原体，难以培养，传统聚合酶链反应（PCR）的检测率差异很大。本文描述了一种基于TaqMan技术的新型定量ompA实时PCR检测方法，用于检测呼吸道样本中的肺炎衣原体，并将其在灵敏度和可重复性方面的性能与四种已发表的传统PCR方法进行了比较（一种针对克隆的PstI片段的单步PCR；两种巢式PCR，一种针对16S核糖体RNA（rRNA）基因，随后进行杂交，另一种针对ompA基因；以及一种同样针对16S rRNA基因的降落酶延时释放（TETR）PCR）。两种基于ompA的PCR显示出最佳的分析灵敏度。所有五种检测方法都能从加标的痰液中检测到更低的靶标水平，16S rRNA检测方法的表现优于基于ompA的巢式PCR（16S rRNA TETR PCR和16S rRNA巢式PCR分别在四个重复样本中的四个和两个中检测到10⁻⁶包涵体形成单位[IFU]）。总体而言，基于ompA的实时检测方法对所有测试重复样本产生的阳性结果最一致，低至10⁻⁶ IFU。在至少一种检测方法测试的四个重复样本中，45份患者痰液标本中有8份（18%）至少在一次检测中肺炎衣原体DNA呈阳性。不考虑检测结果的分析灵敏度或可重复性，16S rRNA TETR检测、基于PstI的单步PCR、基于ompA的实时PCR、基于ompA的巢式降落PCR和基于16S rRNA的巢式PCR检测到的肺炎衣原体DNA阳性痰液标本数量分别为4份、3份、2份、2份和1份。然而，阳性结果的总体一致性率较低。只有一份细胞培养阳性的痰液标本在五种检测方法中的四种检测中呈阳性（16个重复样本中的14个；平均循环阈值，25；痰液中10⁸个颗粒/毫升）。37份标本在所有测试重复样本中通过所有五种检测方法均为肺炎衣原体阴性，所有阴性对照也是如此（每个测试组n = 65至100）。通过实时PCR或基于16S rRNA的巢式检测未检测到PCR抑制剂。我们证实，检测肺炎衣原体的检测方法的分析灵敏度不一定能预测其在痰液中检测靶标的能力。本文所述的基于ompA的实时TaqMan PCR这样一种定量、快速且易于操作的诊断方法可能会提高呼吸道样本中肺炎衣原体的检测率。
