Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification.
10.12116/j.issn.1004-5619.2021.510802
- Author:
Sun-Xiang WEI
1
;
Hui-Xiang CHEN
1
;
Sheng HU
2
;
Yi-Xia ZHAO
2
;
Hui-Xia SHI
2
;
Zhe WANG
2
;
Wen LI
2
;
An-Quan JI
2
;
Qi-Fan SUN
2
Author Information
1. School of Forensic Medicine, Shanxi Medical University, Taiyuan 030001, China.
2. Key Laboratory of Forensic Genetics, Ministry of Public Security, National Engineering Laboratory for Forensic Science, Institute of Forensic Science, Beijing 100038, China.
- Publication Type:Journal Article
- Keywords:
droplet digital polymerase chain reaction (ddPCR);
forensic genetics;
miR-888;
miR-891a;
microRNA;
semen
- MeSH:
Female;
Humans;
Body Fluids/chemistry*;
MicroRNAs/analysis*;
Real-Time Polymerase Chain Reaction/methods*;
Saliva/chemistry*;
Semen/chemistry*;
Male
- From:
Journal of Forensic Medicine
2022;38(6):719-725
- CountryChina
- Language:English
-
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 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.
