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

doi:10.12116/j.issn.1004-5619.2021.510802

Abstract

Abstract:

Objective 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%. Conclusion 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.

Key words: forensic genetics, droplet digital polymerase chain reaction （ddPCR）, microRNA, semen, miR-888, miR-891a

CLC Number:

DF795.2

Sun-xiang WEI, Hui-xiang CHEN, Sheng HU, Yi-xia ZHAO, Hui-xia SHI, Zhe WANG, Wen LI, An-quan JI, Qi-fan SUN. Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification[J]. Journal of Forensic Medicine, 2022, 38(6): 719-725.

Figures/Tables 4

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miRNA	信息类型	序列（5'→3'）
miR-888	成熟序列	UACUCAAAAAGCUGUCAGUCA
	逆转录茎环引物	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGACTG
	特异性正向引物	AGGCGCTACTCAAAAAGCTGTC
	通用反向引物	CCAGTGCAGGGTCCGAGGTA
	水解探针	6-FAM-CACTGGATACGACTGACT-MGB
miR-891a	成熟序列	UGCAACGAACCUGAGCCACUGA
	逆转录茎环引物	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGTG
	特异性正向引物	CACGCCTGCAACGAACCT
	通用反向引物	CCAGTGCAGGGTCCGAGGTA
	水解探针	HEX-ATACGACTCAGTGGCTC-MGB

miRNA	信息类型	序列（5'→3'）
miR-888	成熟序列	UACUCAAAAAGCUGUCAGUCA
	逆转录茎环引物	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGACTG
	特异性正向引物	AGGCGCTACTCAAAAAGCTGTC
	通用反向引物	CCAGTGCAGGGTCCGAGGTA
	水解探针	6-FAM-CACTGGATACGACTGACT-MGB
miR-891a	成熟序列	UGCAACGAACCUGAGCCACUGA
	逆转录茎环引物	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGTG
	特异性正向引物	CACGCCTGCAACGAACCT
	通用反向引物	CCAGTGCAGGGTCCGAGGTA
	水解探针	HEX-ATACGACTCAGTGGCTC-MGB

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