Schabereiter-Gurtner C, Hufnagl P, Sonvilla G, Selitsch B, Rotter M L, Makristathis A, Hirschl A M
Department of Clinical Microbiology, Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Vienna, Austria.
Clin Microbiol Infect. 2008 May;14(5):480-6. doi: 10.1111/j.1469-0691.2008.01962.x. Epub 2008 Mar 4.
A novel HybProbe real-time LightCycler PCR assay was developed for confirmation of Neisseria gonorrhoeae in samples positive according to the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR assay. The new assay amplifies 375 bp of the N. gonorrhoeae 16S rRNA gene and includes an internal amplification control introduced during DNA purification. The assay had 100% specificity because of the high specificity of the HybProbes and primers. Other Neisseria spp. failed to generate positive crossing-point values and melting peaks. The analytical sensitivity for N. gonorrhoeae DNA was 0.5 fg/PCR, corresponding to 0.3 CFU/PCR. Sensitivity was not impaired in the presence of higher DNA concentrations (>or=1000-fold) from Neisseria spp. other than N. gonorrhoeae. The sensitivity was similar to that reported for the COBAS AMPLICOR assay with cervical swab samples. To assess its clinical applicability as a confirmatory test, 38 (2.9%) of 1313 swabs that were positive according to the COBAS AMPLICOR assay were tested using the new in-house assay and the commercially available GenFlow Neisseria test. Twenty-one samples negative according to COBAS AMPLICOR also underwent confirmatory testing. Both confirmatory tests yielded identical results; the 21 negative samples remained negative, and only 11 (28.9%) of the samples positive according to COBAS AMPLICOR were positive after retesting, suggesting a low prevalence (0.84%) of N. gonorrhoeae infection in the study population. These data suggest that the novel real-time PCR assay is an excellent and easy to interpret confirmatory test for the existing COBAS AMPLICOR assay for N. gonorrhoeae.
针对COBAS AMPLICOR沙眼衣原体/淋病奈瑟菌PCR检测呈阳性的样本，开发了一种新型HybProbe实时荧光定量PCR检测方法以确认淋病奈瑟菌。该新检测方法扩增淋病奈瑟菌16S rRNA基因的375 bp片段，并在DNA纯化过程中引入了内部扩增对照。由于HybProbes和引物具有高特异性，该检测方法具有100%的特异性。其他奈瑟菌属未能产生阳性交叉点值和熔解峰。淋病奈瑟菌DNA的分析灵敏度为0.5 fg/PCR，相当于0.3 CFU/PCR。在存在高于淋病奈瑟菌的其他奈瑟菌属更高DNA浓度（≥1000倍）的情况下，灵敏度不受影响。该灵敏度与宫颈拭子样本的COBAS AMPLICOR检测报告的灵敏度相似。为了评估其作为确证试验的临床适用性，使用新的内部检测方法和市售的GenFlow奈瑟菌检测方法对1313份根据COBAS AMPLICOR检测呈阳性的拭子中的38份（2.9%）进行了检测。21份根据COBAS AMPLICOR检测呈阴性的样本也进行了确证检测。两种确证检测结果相同；21份阴性样本仍为阴性，根据COBAS AMPLICOR检测呈阳性的样本中只有11份（28.9%）在重新检测后呈阳性，这表明研究人群中淋病奈瑟菌感染的患病率较低（0.84%）。这些数据表明，这种新型实时PCR检测方法是现有COBAS AMPLICOR淋病奈瑟菌检测方法的一种出色且易于解释的确证检测方法。
