Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi, USA.
J Clin Microbiol. 2013 Jul;51(7):2033-9. doi: 10.1128/JCM.00196-13. Epub 2013 Apr 17.
Health care-associated infections with methicillin-resistant Staphylococcus aureus (MRSA) contribute to significant hospitalization costs. We report here a droplet digital PCR (ddPCR) assay, which is a next-generation emulsion-based endpoint PCR assay for high-precision MRSA analysis. Reference cultures of MRSA, methicillin-susceptible S. aureus (MSSA), and confounders were included as controls. Copan swabs were used to sample cultures and collect specimens for analysis from patients at a large teaching hospital. Swab extraction and cell lysis were accomplished using magnetic-driven agitation of silica beads. Quantitative PCR (qPCR) (Roche Light Cycler 480) and ddPCR (Bio-Rad QX100 droplet digital PCR system) assays were used to detect genes for the staphylococcal protein SA0140 (SA) and the methicillin resistance (mecA) gene employing standard TaqMan chemistries. Both qPCR and ddPCR assays correctly identified culture controls for MRSA (76), MSSA (12), and confounder organisms (36) with 100% sensitivity and specificity. Analysis of the clinical samples (211 negative and 186 positive) collected during a study of MRSA nasal carriage allowed direct comparison of the qPCR and ddPCR assays to the Cepheid MRSA GeneXpert assay. A total of 397 clinical samples were examined in this study. Cepheid MRSA GeneXpert values were used to define negative and positive samples. Both the qPCR and ddPCR assays were in good agreement with the reference assay. The sensitivities for the qPCR and ddPCR assays were 96.8% (95% confidence interval [CI], 93.1 to 98.5%) and 96.8% (95% CI, 93.1 to 98.5%), respectively. Both the qPCR and ddPCR assays had specificities of 91.9% (95% CI, 87.5 to 94.9%) for qPCR and 91.0% (95% CI, 86.4 to 94.2%) for ddPCR technology.
耐甲氧西林金黄色葡萄球菌（MRSA）引起的医疗保健相关感染导致了大量的住院费用。我们在此报告一种液滴数字 PCR（ddPCR）检测方法，它是一种基于下一代乳液的终点 PCR 检测方法，用于高度精确的 MRSA 分析。MRSA、甲氧西林敏感金黄色葡萄球菌（MSSA）和混杂因素的参考培养物被用作对照。使用 Copan 拭子从一家大型教学医院的患者身上采集培养物样本和用于分析的标本。使用磁驱动搅拌硅珠完成拭子提取和细胞裂解。采用定量 PCR（qPCR）（罗氏 Light Cycler 480）和 ddPCR（Bio-Rad QX100 液滴数字 PCR 系统）检测方法，采用标准 TaqMan 化学法检测金黄色葡萄球菌蛋白 SA0140（SA）和耐甲氧西林基因（mecA）基因。qPCR 和 ddPCR 检测方法均能正确识别 76 株 MRSA、12 株 MSSA 和 36 株混杂菌的培养物对照，具有 100%的敏感性和特异性。在对 186 例 MRSA 鼻携带的临床样本（211 例阴性和 186 例阳性）的研究中，对 qPCR 和 ddPCR 检测方法与 Cepheid MRSA GeneXpert 检测方法进行了直接比较。这项研究共检测了 397 例临床样本。Cepheid MRSA GeneXpert 值用于定义阴性和阳性样本。qPCR 和 ddPCR 检测方法均与参考检测方法吻合良好。qPCR 和 ddPCR 检测方法的敏感性分别为 96.8%（95%置信区间[CI]，93.1%至 98.5%）和 96.8%（95%CI，93.1%至 98.5%）。qPCR 和 ddPCR 检测方法的特异性分别为 91.9%（95%CI，87.5%至 94.9%）和 91.0%（95%CI，86.4%至 94.2%）。
