Preliminary Exploration of microRNA Body Fluid Identification Panel Based on Droplet Digital PCR

doi:10.3969/j.issn.1671-2072.2025.06.004

Abstract

Abstract: Objective To investigate the detection efficacy and application potential of a microRNA (miRNA) five forensic body fluid identification system, originally constructed using real-time fluorescence quantitative PCR(qPCR), when adapted to the droplet digital PCR (ddPCR) platform. Methods Stem-loop reverse transcription primers and specific PCR primers were designed to establish a ddPCR detection system targeting fourteen body fluid-specific miRNA markers. Following data normalization and transformation, miRNA expression patterns and abundance obtained from ddPCR and qPCR platforms were comparatively analyzed. A multi-class support vector machine (MSVM) classifier was employed to infer the source of body fluid samples. Results A ddPCR detection system utilizing EvaGreen dye was successfully developed for quantifying fourteen body fluid-specific miRNA markers. The comparative analysis indicated good agreement between ddPCR absolute quantification and qPCR relative quantification platforms, with a Pearson correlation coefficient (r) of 0.9. However, the MSVM model developed based on qPCR data demonstrated suboptimal classification accuracy when directly applied to ddPCR data. Conclusion The study preliminarily validated the performance and demonstrated considerable application potential of the ddPCR platform for forensic body fluid identification using miRNA markers. However, further optimization of the detection system and development of a ddPCR-specific body fluid classification model are still required.

Key words: forensic genetics, body fluid identification, microRNA (miRNA), droplet digital PCR (ddPCR), multi-class support vector machine (MSVM)

摘要： 目的探讨基于实时荧光定量PCR（real-time fluorescence quantitative PCR，qPCR）构建的微RNA（microRNA，miRNA）法医学5种体液识别体系在微滴式数字PCR（droplet digital PCR，ddPCR）上的检测效能与应用潜力。方法通过设计茎环结构的逆转录引物及特异性PCR引物，构建14个体液特异性miRNA标记的ddPCR检测体系，数据转化后对比分析miRNA 标记在ddPCR和qPCR上的表达模式和丰度，并基于多类别支持向量机（multi-class support vector machine，MSVM）进行体液来源推断。结果成功构建了14个体液特异性miRNA标记的ddPCR染料法检测体系；一致性分析显示两个定量平台的表达水平检测存在较高的一致性（皮尔逊相关系数r为0.9）；但基于qPCR开发的MSVM溯源模型应用于ddPCR数据上的准确率欠佳。结论初步探索了体液特异性miRNA标记体系在ddPCR平台上的效能，显示出较强的应用潜力，但仍需进一步优化体系并开发针对ddPCR的体液溯源模型。

关键词: 法医物证学, 体液溯源, 微RNA, 微滴式数字PCR, 多类别支持向量机

CLC Number:

DF795.2

YAN Zichen, LI Suyu, LIU Jing, CONG Bin, WANG Zheng. Preliminary Exploration of microRNA Body Fluid Identification Panel Based on Droplet Digital PCR[J]. Chinese Journal of Forensic Sciences, 2025(6): 31-37.

鄢子辰, 李苏豫, 刘京, 丛斌, 王正. 数字PCR在microRNA识别体液类型中的初步研究[J]. 中国司法鉴定, 2025(6): 31-37.

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