Objectives: To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification.
Methods: The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained.
Results: There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/μL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/μL, and the discrimination accuracy was 100%.
Conclusions: In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.
目的: 利用微滴式数字PCR（droplet digital PCR，ddPCR）技术建立可同时检测miR-888和miR-891a的体系，并评估其在精液鉴定中的应用价值。方法: 通过设计不同荧光修饰的报告基团的水解探针实现对miR-888和miR-891a的双重ddPCR检测，对5种体液（外周血、月经血、精液、唾液、阴道分泌液）共75份样本进行检测，采用Mann-Whitney U检验进行差异性分析，通过ROC曲线分析评估miR-888和miR-891a区分精液的能力并获得鉴别的最佳截断值。结果: 该体系双重检测与单独检测结果差异无统计学意义，检测灵敏度可达到0.1 ng总RNA，批内与批间变异系数均小于15%。经双重ddPCR检测，精液中miR-888和miR-891a的表达量均高于其他体液。经ROC曲线分析，miR-888的AUC为0.976，最佳截断值为2.250 copies/µL，判别正确率为97.33%；miR-891a的AUC为1.000，最佳截断值为1.100 copies/µL，判别正确率为100%。结论: 本研究成功建立了双重ddPCR检测miR-888和miR-891a的方法，体系的稳定性和重复性良好，可用于精液鉴定，miR-888和miR-891a均具有较高的精液鉴别能力，且miR-891a的判别正确率更高。.
Keywords: droplet digital polymerase chain reaction (ddPCR); forensic genetics; miR-888; miR-891a; microRNA; semen.