Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Brussels, Belgium.
J Mol Diagn. 2011 Mar;13(2):206-12. doi: 10.1016/j.jmoldx.2010.10.007.
We evaluated a previously described quantitative real-time PCR (qPCR) for quantifying and differentiating Ureaplasma parvum and U. urealyticum. Because of nonspecific reactions with Staphylococcus aureus DNA in the U. parvum PCR, we developed a modified qPCR and designed new primers. These oligonucleotides eradicated cross-reactions, indicating higher specificity. The detection limits of the qPCR were determined at 1 and 3 colony-forming units/ml for U. parvum and U. urealyticum, respectively. The quantification limits of the assay for both Ureaplasma species ranged from 2.10(6) to 2.10(1) copy numbers per PCR. A total of 300 patient samples obtained from the lower genital tract were tested with this newly designed qPCR assay and compared with culture results. Of the samples, 132 (44.0%) were culture positive, whereas 151 (50.3%) tested positive using qPCR. The U. parvum and U. urealyticum species were present in 79.5% and 12.6% of the qPCR-positive samples, respectively. Both species were found in 7.9% of those samples. Quantification of U. parvum and U. urealyticum in the samples ranged from less than 2.5 × 10(3) to 7.4 × 10(7) copies per specimen. In conclusion, the modified qPCR is a suitable method for rapid detection, differentiation, and quantification of U. parvum and U. urealyticum.
我们评估了一种先前描述的定量实时 PCR(qPCR)，用于定量和区分微小脲原体和脲原体。由于 U. parvum PCR 与金黄色葡萄球菌 DNA 的非特异性反应，我们开发了一种改良的 qPCR 并设计了新的引物。这些寡核苷酸消除了交叉反应，表明更高的特异性。qPCR 的检测限分别为 U. parvum 和 U. urealyticum 的 1 和 3 个菌落形成单位/ml。该测定对两种脲原体的定量限范围分别为 2.10(6)至 2.10(1)个拷贝数/PCR。总共用这种新设计的 qPCR 检测了从下生殖道获得的 300 个患者样本，并与培养结果进行了比较。在这些样本中，132 个（44.0%）为培养阳性，而 151 个（50.3%）使用 qPCR 检测为阳性。qPCR 阳性样本中分别有 79.5%和 12.6%存在微小脲原体和脲原体。在这些样本中，7.9%同时存在这两种病原体。样本中 U. parvum 和 U. urealyticum 的定量范围从小于 2.5×10(3)到 7.4×10(7)个拷贝/标本。总之，改良的 qPCR 是一种快速检测、区分和定量微小脲原体和脲原体的合适方法。
