Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy and School of Medicine 06018, Ankara, Turkey.
Diagn Microbiol Infect Dis. 2010 Apr;66(4):349-55. doi: 10.1016/j.diagmicrobio.2009.11.010.
We describe here a 1-step, triplex real-time polymerase chain reaction (PCR) assay for the detection and identification of staphylococci directly from signal-positive blood culture bottles containing Gram-positive cocci in clusters (GPCC). The triplex assay targeted and detected tuf, nuc, and mecA genes in a single tube and had a detection limit of 10(5) CFU/mL for each gene target. A total of 341 GPCC-positive blood culture bottles were collected between November 12, 2008, and August 11, 2009. Among them, 230 methicillin-resistant coagulase-negative staphylococci (CoNS), 54 methicillin-susceptible CoNS, 22 methicillin-resistant Staphylococcus aureus, 22 methicillin-susceptible S. aureus, and 13 nonstaphylococci species were identified by conventional methods. The results obtained by triplex assay were in agreement with those of conventional methods for tuf (99.7%), nuc (100.0%), and mecA (99.1%), respectively. The triplex assay was found to have sensitivities of 99.7%, 100%, and 99.2% and specificities of 100%, 100%, and 98.7%, respectively, for the tuf, nuc, and mecA gene targets. The triplex real-time PCR assay accurately detects and identifies staphylococci directly from positive blood cultures without nucleic acid extraction prior to amplification.
我们在此描述了一种一步法三重实时聚合酶链反应（PCR）检测法，可直接从革兰阳性球菌呈簇状（GPCC）的信号阳性血培养瓶中检测和鉴定葡萄球菌。该三重检测法靶向并检测 tuf、nuc 和 mecA 基因，在单个管中检测限为每个基因靶标 10（5）CFU/mL。总共收集了 341 个 GPCC 阳性血培养瓶，时间为 2008 年 11 月 12 日至 2009 年 8 月 11 日。其中，230 株耐甲氧西林凝固酶阴性葡萄球菌（CoNS）、54 株甲氧西林敏感 CoNS、22 株耐甲氧西林金黄色葡萄球菌、22 株甲氧西林敏感金黄色葡萄球菌和 13 株非葡萄球菌种通过常规方法鉴定。三重检测法在 tuf（99.7%）、nuc（100.0%）和 mecA（99.1%）方面的检测结果分别与常规方法一致。该三重检测法对 tuf、nuc 和 mecA 基因靶标的灵敏度分别为 99.7%、100%和 99.2%，特异性分别为 100%、100%和 98.7%。该三重实时 PCR 检测法可在无需扩增前核酸提取的情况下，直接从阳性血培养物中准确检测和鉴定葡萄球菌。
