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应用双液滴数字 PCR 检测 miR-888 和 miR-891a 在精液鉴定中的应用。

Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification.

机构信息

School of Forensic Medicine, Shanxi Medical University, Taiyuan 030001, China.

Key Laboratory of Forensic Genetics, Ministry of Public Security, National Engineering Laboratory for Forensic Science, Institute of Forensic Science, Beijing 100038, China.

出版信息

Fa Yi Xue Za Zhi. 2022 Dec 25;38(6):719-725. doi: 10.12116/j.issn.1004-5619.2021.510802.

Abstract

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 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.

摘要

目的

建立一种通过数字液滴 PCR(ddPCR)同时检测 miR-888 和 miR-891a 的系统,并评估其在精液鉴定中的应用价值。

方法

设计具有不同荧光修饰报告基团的水解探针,通过双 ddPCR 实现 miR-888 和 miR-891a 的检测。检测 75 例 5 种体液(外周血、月经血、精液、唾液和阴道分泌物)样本,采用 Mann-Whitney U 检验进行差异分析。通过 ROC 曲线分析评价 miR-888 和 miR-891a 的精液鉴别能力,并获得最佳截断值。

结果

该系统中双探针检测与单探针检测无显著差异。检测灵敏度可达 0.1ng 总 RNA,内、批间变异系数均小于 15%。双 ddPCR 检测到精液中 miR-888 和 miR-891a 的表达水平均高于其他体液。ROC 曲线分析显示,miR-888 的 AUC 为 0.976,最佳截断值为 2.250 拷贝/μL,鉴别准确率为 97.33%;miR-891a 的 AUC 为 1.000,最佳截断值为 1.100 拷贝/μL,鉴别准确率为 100%。

结论

本研究成功建立了一种双 ddPCR 检测 miR-888 和 miR-891a 的方法。该系统具有良好的稳定性和重复性,可用于精液鉴定。miR-888 和 miR-891a 均具有较高的精液鉴别能力,miR-891a 的鉴别准确率更高。

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