Clinical Research Department, London School of Hygiene and Tropical Medicine, London, United Kingdom.
J Clin Microbiol. 2013 Jul;51(7):2195-203. doi: 10.1128/JCM.00622-13. Epub 2013 May 1.
Droplet digital PCR (ddPCR) is an emulsion PCR process that performs absolute quantitation of nucleic acids. We developed a ddPCR assay for Chlamydia trachomatis infections and found it to be accurate and precise. Using PCR mixtures containing plasmids engineered to include the PCR target sequences, we were able to quantify with a dynamic range between 0.07 and 3,160 targets/μl (r(2) = 0.9927) with >95% confidence. Using 1,509 clinical conjunctival swab samples from a population in which trachoma is endemic in Guinea Bissau, we evaluated the specificity and sensitivity of the quantitative ddPCR assay in diagnosing ocular C. trachomatis infections by comparing the performances of ddPCR and the Roche Amplicor CT/NG test. We defined ddPCR tests as positive when we had ≥95% confidence in a nonzero estimate of target load. The sensitivity of ddPCR against Amplicor was 73.3% (95% confidence interval [CI], 67.9 to 78.7%), and specificity was 99.1% (95% CI, 98.6 to 99.6%). Negative and positive predictive values were 94.6% (95% CI, 93.4 to 95.8%) and 94.5% (95% CI, 91.3 to 97.7%), respectively. Based on Amplicor CT/NG testing, the estimated population prevalence of C. trachomatis ocular infection was ∼17.5%. Receiver-operator curve analysis was used to select critical cutoff values for use in clinical settings in which a balance between higher sensitivity and specificity is required. We concluded that ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections.
数字液滴 PCR（ddPCR）是一种乳液 PCR 过程，可对核酸进行绝对定量。我们开发了一种用于沙眼衣原体感染的 ddPCR 检测方法，发现其准确且精确。使用包含经工程设计以包含 PCR 靶序列的质粒的 PCR 混合物，我们能够以 0.07 至 3,160 个靶标/μl 的动态范围进行定量（r² = 0.9927），置信度超过 95%。我们使用来自几内亚比绍流行沙眼的人群的 1,509 例临床结膜拭子样本，通过比较 ddPCR 和罗氏 Amplicor CT/NG 检测在诊断眼部沙眼衣原体感染方面的性能，评估了定量 ddPCR 检测的特异性和敏感性。当我们对靶标负荷的非零估计有≥95%的置信度时，我们将 ddPCR 检测定义为阳性。ddPCR 对 Amplicor 的敏感性为 73.3%（95%置信区间 [CI]，67.9 至 78.7%），特异性为 99.1%（95%CI，98.6 至 99.6%）。阴性和阳性预测值分别为 94.6%（95%CI，93.4 至 95.8%）和 94.5%（95%CI，91.3 至 97.7%）。根据 Amplicor CT/NG 检测，沙眼衣原体眼部感染的估计人群患病率约为 17.5%。使用接收者操作特征曲线分析选择了用于需要在诊断眼部沙眼衣原体感染时平衡更高敏感性和特异性的临床环境中的临界截断值。我们得出结论，ddPCR 是一种有效的诊断技术，适合于研究和临床使用。
