一步法mRNA体液鉴定体系的建立及应用

Establishment and Application of One-step mRNA Body Fluid Identification System

为实现常见法医体液类型的快速鉴定,本研究挑选出10种具有体液表达特异性的mRNA及3种管家基因,并通过筛选一步法RT-PCR试剂盒、调整引物浓度、优化反应温度和时间,成功建立了一种可同时检测13种mRNA的一步法RT-PCR实验体系。使用该体系对144份样本(包括外周血、月经血、精液、唾液、阴道分泌液)进行检测并分析,多数目标基因特异性良好,检测时长在3 h以内,对外周血样本检测灵敏度可达0.01 ng RNA,可在混合样本中检测到混合成分的目标mRNA。虽然室温保存3~4年样本会有标记未检测到,但本实验体系具有操作简单、混合样本鉴别能力强、耗时短的优势,具有良好的实战应用前景。

Body fluid stains are common biological materials at crime scenes.The accurate determination of their tissue sources can help with crime scene reconstruction,case nature determination and trial.The analysis of cell-specific mRNA expression has been proposed as promising method for the identification of body fluids.Conventional strategy of mRNA profiling requires reverse transcription,PCR amplification,and electrophoresis.The one-step RT-PCR detection technology can complete reverse transcription and PCR of mRNA in one reaction,which can reduce experimental time and simplify experimental operations.In this study,we subjected the one-step multiplex reverse transcription PCR strategy to mRNA profiling with the inclusion of 10 tissue specific biomarkers in the F13plex system targeting peripheral blood(HBA,HBB),menstrual blood(MMP7,MMP10),vaginal secretion(HBD1,CYP2B7P),saliva(STATH,HTN3)and semen(PRM2,SEMG1),and 3 housekeeping genes(ACTB,GAPDH and RPL19).We verified the system’s specificity,sensitivity,and ability to detect mixed and aged samples.In terms of specificity,most of the selected genes had good specificity,but there were some cross-reactions that were hard to avoid.In order to ensure the accuracy of identification,we determined that the target body fluid was contained only when the three housekeeping genes and two specific mRNA markers were simultaneously detected.In terms of sensitivity,we found that different types of samples had different sensitivities.For example,when using 10 ng RNA for vaginal secretions and menstrual blood samples,some specific target genes were not detected and could not be correctly determined;for blood samples,even when 0.01 ng RNA was used,the RFU value of the target gene is still above 10000.However,there are only a small amount of test materials in actual cases;it is difficult to quantify the extracted RNA.For five kinds of body fluids,2μL of RNA extracted from a 1 mm2 sample could all detect housekeeping genes and corresponding target genes,and the correct body fluid could be determined(Except 1 vaginal secretion sample).The target genes of the mixed components were detected in all 16 mixed samples,and correct mixed component determinations could be made,proving the system’s good ability to identify mixed samples.Almost all housekeeping genes could be detected in 14 aged samples,but only 7 were correctly identified.Because no specific target genes were detected in the remaining samples,correct determinations could not be made.Consequently,the system needs to be further optimized.For HBD1 and MMP7 with poor specificity,other vaginal secretion and menstrual blood-specific genes will be screened and verified for replacement.The instability of mRNA results in relatively poor test results for aged samples.In practical applications,other genetic markers with better stability should be used to determine the results.In general,lots of studies have been demonstrated the usability of mRNA profiling to the identification of forensic relevant body fluid.According to the comprehensive assessment of the onestep RT-PCR strategy in this study,the one-step profiling assays can be a reliable and economical method for the simplified,accurate,and simultaneous analysis of tissue-specific biomarkers for the discrimination of body fluid origin.It shows good application potential in forensic body fluid identification work.