用于法医体液鉴定的多路实时定量PCR分析的部分验证

IF 1.9 4区 医学 Q2 MEDICINE, LEGAL
Courtney Lynch , Rachel Fleming
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引用次数: 1

摘要

使用信使核糖核酸(信使核糖核酸)进行确认性体液识别是一种公认的技术,可以解决传统检测中遇到的问题,例如灵敏度、特异性差,以及缺乏对所有感兴趣体液的可用检测。十多年来,端点逆转录聚合酶链式反应(RT-PCR)检测已被用于此类目的的法医案例工作。然而,与实时定量RT-PCR(RT-qPCR)相比,终点RT-PCR具有较低的灵敏度、准确度和线性动态范围。本研究详细介绍了验证性RT-qPCR测定的多路复用和部分验证。我们之前已经描述了一系列体液靶标的新测定方法,并确定了用于扩增它们的最佳商业试剂盒。在此,进行多路复用以形成三种测定:循环血液(SLC4A1)和月经液(STC1)、唾液(HTN3)和阴道物质(CYP2B7P)、精子(PRM1)和精液(KLK2),所有这些都包括合成的内部对照RNA。多路复用测定的部分验证包括MIQE指南、ISO要求和SWGDAM指南。使用受试者操作特征(ROC)曲线,每个标记物与无信息测定显著不同,最佳截止值均在35个周期以上。所有测定都显示出宽的LDR(范围从3到5个对数,大多数R2>;0.99)和高精度(大多数平均CV<;1%)。STC1在高CT值的血液、精液和阴道物质中显示出一些散发性表达。CYP2B7P在精液和血液中表现出脱靶表达。敏感性近似为:;唾液:全颊拭子的1/1000稀释液,循环血液:0.01-0.1µL血液,经液:全经拭子的万分之一稀释液,精子:0.001µL精液,精液:0.01µL精液和阴道材料:全阴道拭子的1/1000稀释液。总共测试了16种模拟体液提取物混合物和18种拭子混合物,在每个特定截止点以下,目标标记物的检测率分别为100%和99%。一些含有大量血液和精液的混合物显示出CYP2B7P的脱靶表达。还证明了概率模型在RT-qPCR数据中的成功应用。进一步的工作将涉及全面的开发验证。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Partial validation of multiplexed real-time quantitative PCR assays for forensic body fluid identification

Confirmatory body fluid identification using messenger RNA (mRNA) is a well-established technique to address issues encountered with conventional testing – such as poor sensitivity, specificity, and a lack of available tests for all body fluids of interest. For over a decade, endpoint reverse-transcription polymerase chain reaction (RT-PCR) assays have been used in forensic casework for such purposes. However, in comparison with real-time quantitative RT-PCR (RT-qPCR), endpoint RT-PCR has lower sensitivity, precision, and linear dynamic range.

This research details the multiplexing and partial validation of confirmatory RT-qPCR assays. We have previously described novel assays for a range of body fluid targets and identified an optimal commercial kit for their amplification. Here, multiplexing was undertaken to form three assays: circulatory blood (SLC4A1) and menstrual fluid (STC1), saliva (HTN3) and vaginal material (CYP2B7P), and spermatozoa (PRM1) and seminal fluid (KLK2), all including a synthetic internal control RNA.

Partial validation of the multiplexed assays incorporated the MIQE guidelines, ISO requirements, and SWGDAM guidelines. Using receiver operating characteristic (ROC) curves, each marker was significantly different from an uninformative assay and optimal cut-offs were all above 35 cycles. All assays showed a wide LDR (ranging from 3 to 5 logs with most R2 > 0.99), and high precision (most mean CV < 1 %). STC1 showed some instances of sporadic expression in blood, semen, and vaginal material at high CT values. CYP2B7P showed off-target expression in semen and blood. The sensitivities were approximated as; saliva: 1 in 1,000 dilution of a whole buccal swab, circulatory blood: 0.01–0.1 µL blood, menstrual fluid: 1 in 10,000 dilution of a whole menstrual swab, spermatozoa: 0.001 µL semen, seminal fluid: 0.01 µL semen, and vaginal material: 1 in 1,000 dilution of a whole vaginal swab.

A total of 16 mock body fluid extract mixtures and 18 swab mixtures were tested and had 100% and 99% detection of target markers below each specific cut-off, respectively. Some mixtures containing high volumes of blood and semen showed off-target CYP2B7P expression. The successful application of a probabilistic model to the RT-qPCR data was also demonstrated. Further work will involve full developmental validation.

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来源期刊
Science & Justice
Science & Justice 医学-病理学
CiteScore
4.20
自引率
15.80%
发文量
98
审稿时长
81 days
期刊介绍: Science & Justice provides a forum to promote communication and publication of original articles, reviews and correspondence on subjects that spark debates within the Forensic Science Community and the criminal justice sector. The journal provides a medium whereby all aspects of applying science to legal proceedings can be debated and progressed. Science & Justice is published six times a year, and will be of interest primarily to practising forensic scientists and their colleagues in related fields. It is chiefly concerned with the publication of formal scientific papers, in keeping with its international learned status, but will not accept any article describing experimentation on animals which does not meet strict ethical standards. Promote communication and informed debate within the Forensic Science Community and the criminal justice sector. To promote the publication of learned and original research findings from all areas of the forensic sciences and by so doing to advance the profession. To promote the publication of case based material by way of case reviews. To promote the publication of conference proceedings which are of interest to the forensic science community. To provide a medium whereby all aspects of applying science to legal proceedings can be debated and progressed. To appeal to all those with an interest in the forensic sciences.
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