微滴式数字 PCR 定量检测伯氏考克斯氏体方法的建立及应用

王亚鹏 ¹，曲瑶 ²，张皓博 ³，李嘉琪 ⁴，焉鑫 ⁴，孙淑芳 ³，孙翔翔 ⁴，欧阳康 ⁵

Abstract

为建立伯氏考克斯氏体（Coxiella burnetii, Cb）微滴式数字 PCR（Droplet Digital PCR, ddPCR）检测方法，研究以 Cb IS1111基因为靶基因，针对基因保守区域设计特异性引物和探针，优化反应条件构建 ddPCR 检测方法，并评估该方法的特异性、敏感性及重复性。结果显示，该方法最低检出浓度为0.52 copies/μL，敏感性较高；与动物布鲁氏菌、牛结核分枝杆菌、土拉杆菌、金黄色葡萄球菌和沙门氏菌无交叉反应，特异性强；组内重复和组间重复变异系数均小于10 %，重复性好。采用本方法检测 42 份疑似感染临床样品，检测结果与荧光 PCR 鉴定结果一致。建立的 ddPCR 方法特异性强、灵敏度高、重复性与准确性良好，可用于 Cb 快速检测和精准定量，对 Cb 感染防控具有重要意义。

In order to establish a Droplet Digital PCR (ddPCR) detection method for Coxiella burnetii (Cb), this study designed specific primers and probes targeting the conserved region of the Cb IS1111 gene and optimized reaction conditions. A ddPCR detection assay was established, and its specificity, sensitivity and repeatability were evaluated. The results showed that the limit of detection was 0.52 copies/μL, reflecting high sensitivity. No cross-reactivity was observed with animal Brucella, Mycobacterium bovis, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella, presenting favorable specificity. Both intra-assay and inter-assay coefficients of variation were lower than 10 %. A total of 42 suspected clinical samples were tested, and the results were consistent with those obtained by fluorescent PCR. The established ddPCR method features high specificity, sensitivity, repeatability and accuracy, and is applicable to rapid detection and absolute quantification of Cb, which facilitates prevention and control of Cb infection.