Department of Pediatrics, Sakura Medical Center, Toho University, 564-1 Shimoshizu, Sakura, Chiba, Japan,
J Infect Chemother. 2013 Dec;19(6):1087-92. doi: 10.1007/s10156-013-0630-9. Epub 2013 Jun 17.
It is not known that saliva is useful to diagnose Mycoplasma pneumoniae (M. pneumoniae) infection by PCR. We evaluated prospectively whether crude saliva samples without the DNA extraction process were useful for the detection of M. pneumoniae DNA in a locked nucleic acid (LNA) probe-based real-time PCR assay. Fifty-one clinical specimens (29 sputum, 22 saliva) that were positive by conventional M. pneumoniae-specific PCR were evaluated in this study. We designed an LNA probe-based real-time PCR that could discriminate the mutant strain (A2063G mutation) from the wild-type strain. All the 51 samples treated with DNA extraction were positive using the LNA probe-based real-time PCR. The results of the real-time PCR with DNA extraction were consistent with the sequence analysis. Of the 51 samples without DNA extraction, on the other hand, 41 (80.4%) were positive by real-time PCR. Of 29 sputum samples without DNA extraction, 23 (79.3%) were positive by real-time PCR; of the 22 saliva samples without DNA extraction, 18 (81.8%) were positive by real-time PCR. There was a statistically significant difference in the amplified DNA levels with extraction between the direct real-time PCR-positive samples (mean ± SD, 7.5 ± 1.6 log copies/ml) and PCR-negative samples (4.2 ± 0.8 log copies/ml) (P < 0.001). Saliva was useful for a template for PCR as well as sputum. In addition, crude samples were useful for real-time PCR when the samples had medium or high DNA levels. However, samples with low DNA levels sometimes showed false-negative results in direct real-time PCR.
目前尚不清楚唾液是否可通过 PCR 用于诊断肺炎支原体 (Mycoplasma pneumoniae, M. pneumoniae) 感染。我们前瞻性评估了未经 DNA 提取过程的粗唾液样本是否可用于基于锁核酸 (locked nucleic acid, LNA) 探针的实时 PCR 检测中检测 M. pneumoniae DNA。本研究评估了 51 份临床标本（29 份痰，22 份唾液），这些标本经常规 M. pneumoniae 特异性 PCR 均为阳性。我们设计了一种基于 LNA 探针的实时 PCR 检测方法，该方法可区分突变株（A2063G 突变）和野生型株。所有用 LNA 探针进行实时 PCR 检测的 51 份经 DNA 提取的样本均为阳性。实时 PCR 检测结果与序列分析一致。另一方面，未经 DNA 提取的 51 份样本中，41 份（80.4%）实时 PCR 检测为阳性。未经 DNA 提取的 29 份痰样本中，23 份（79.3%）实时 PCR 检测为阳性；未经 DNA 提取的 22 份唾液样本中，18 份（81.8%）实时 PCR 检测为阳性。有提取和无提取的直接实时 PCR 阳性样本（平均 ± 标准差，7.5 ± 1.6 log 拷贝/ml）与 PCR 阴性样本（4.2 ± 0.8 log 拷贝/ml）的扩增 DNA 水平存在统计学显著差异（P < 0.001）。与痰相比，唾液可作为 PCR 的模板。此外，当样本 DNA 水平为中或高水平时，粗制样本也可用于实时 PCR。然而，当样本 DNA 水平较低时，直接实时 PCR 有时会出现假阴性结果。
