miR-451a和miR-21-5p双重实时荧光定量PCR检测体系的建立及应用

Establishment and Application of a Duplex Real Time Fluorogenic Quantitative PCR Assay System for miR-451a and miR-21-5p

目的为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR(real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法设计适用于miRNA双重检验的相关引物及探针并优化实验体系组分,建立基于TaqMan技术的miRNA双重实时荧光定量PCR检测体系并验证其特异性、灵敏度和可重复性;使用此检测体系对58份不同体液样本中的miR-451a与miR-21-5p进行检测,并借助miR-451a与miR-21-5p的比值鉴定法评估该体系的体液鉴别能力;使用该检测体系样本数据确定的最佳截断值对模拟案件样本进行鉴别。结果优化的检测体系能够实现对血液与非血液、月经血与外周血的100%区分,同时可以实现对模拟案件样本的准确鉴别。结论该双重实时荧光定量PCR检测体系将时间和材料成本均缩短至原来的一半,为后续建立更多重的实时荧光定量PCR检测体系并应用于体液鉴别打下了基础。

Objective Body fluid stains left at crime scenes are frequently trace amounts,while the identification of body fluids through real time fluorogenic quantitative technique often necessitates the repeated detection within the limited sample,as multiple miRNA markers are the basis for the identification.Based on the goal of both the throughput and efficiency improvement of miRNA analysis in trace samples,a duplex real time fluorogenic quantitative PCR assay system was designed to accurately quantify two miRNAs simultaneously,and the system should be further verified by actual sample for the body fluid identification.Methods The duplex real time fluorogenic quantitative PCR system of miR-451a to miR-21-5p was established with specially designed primers and probes,and the concentrations of the primers and probes were both optimized.The specificity,sensitivity and reproducibility of the system were validated,while its capability for body fluid identification was assessed using the miR-451a to miR-21-5p ratio.Results The optimized assay system exhibited excellent specificity and repeatability,with coefficients of variation consistently below 8%for both intra-and inter-batch variability.The amplification efficiency of miR-451a and miR-21-5p reached 71.77%and 74.81%,respectively,with high and relatively consistent results.By utilizing this duplex real time fluorogenic quantitative PCR assay system,a total of 58 body fluid samples were analyzed,exhibiting a discrimination rate of 100%between blood and non-blood samples,as well as between peripheral blood and menstrual blood samples.Moreover,the results,obtained from single real time fluorogenic quantitative PCR assay system and duplex real time fluorogenic quantitative PCR assay system,showed no statistically significant difference with randomly selected blood samples(n=20).Compared to previous single real time fluorogenic quantitative PCR assay system,the sensitivity of duplex real time fluorogenic quantitative PCR assay system exhibited remarkable improvement.A minimum input of only 0.1 ng total RNA was sufficient for accurate detection of peripheral blood and menstrual blood samples,while saliva,semen,and vaginal secretion required only 1 ng total RNA for precise identification purposes.Additionally,the duplex real time fluorogenic quantitative PCR assay system successfully differentiated between different types of body fluids in simulated samples under natural outdoor conditions.Conclusion The duplex real time fluorogenic quantitative PCR assay system effectively reduced both the time and material costs by half compared to the single system,especially suitable for the examination of body fluid stains left at crime scenes,solving the contradiction between the trace amount and the multiple sample volumes demand of repeated real time fluorogenic quantitative PCR.The duplex real time fluorogenic quantitative PCR assay successfully distinguished blood and other body fluid,as well as peripheral blood and menstrual blood samples,which maintains an equivalent capability for body fluid identification with half sample,time and reagent consumption.This system provides an efficient tool for identifying suspicious body fluids,as well as a foundation for more multiplexed real time fluorogenic quantitative PCR assay system research.