一步RT-qPCR高通量精子筛查：改进和重新评估。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry Pub Date : 2024-11-27 DOI:10.1016/j.ab.2024.115727

Seiji Kubo , Keito Amai , Fumitaka Nakano , Jin Tanaka , Hideki Niimi

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引用次数: 0

摘要

精子鉴定在性侵案件中至关重要。虽然显微镜分析是精子检测的金标准，但即使对训练有素的人员来说，这也是一个费力的过程。逆转录-实时定量PCR （RT-qPCR）可以通过检测精子特异性mRNA标记，如鱼精蛋白2 (PRM2)，增强筛选能力。本研究旨在建立一种针对PRM2 mRNA的一步RT-qPCR检测方法。我们的方法能够检测低至0.01 μL的精液，具有很高的特异性，并在模拟病例样本中成功检测到PRM2 mRNA。由于所涉及的工作流程简单，我们的分析需要小于30分钟的RNA提取和小于60分钟的RT-qPCR。我们的分析能够实现高通量精子筛查，并为加强性侵犯案件的工作流程提供了一个有前途的策略。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

High-throughput sperm screening using one-step RT-qPCR: Improvement and re-evaluation

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High-throughput sperm screening using one-step RT-qPCR: Improvement and re-evaluation

Sperm identification is crucial in sexual assault cases. While microscopic analysis is the gold standard for sperm detection, it is a laborious procedure even for trained personnel. Reverse transcription-quantitative real-time PCR (RT-qPCR) can enhance the screening by detecting sperm-specific mRNA markers, such as protamine 2 (PRM2). This study aimed to develop a one-step RT-qPCR assay targeting PRM2 mRNA. Our assay was capable of detecting as low as 0.01 μL of semen with high specificity and demonstrated successful detection of PRM2 mRNA in simulated-case samples. Owing to the simple workflow involved, our assay requires <30 min for RNA extraction and <60 min for RT-qPCR. Our assay enables high-throughput sperm screening and offers a promising strategy for enhancing the workflow of sexual assault cases.

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来源期刊

Analytical biochemistry 生物-分析化学

CiteScore

5.70

自引率

0.00%

发文量

283

审稿时长

44 days

期刊介绍： The journal''s title Analytical Biochemistry: Methods in the Biological Sciences declares its broad scope: methods for the basic biological sciences that include biochemistry, molecular genetics, cell biology, proteomics, immunology, bioinformatics and wherever the frontiers of research take the field. The emphasis is on methods from the strictly analytical to the more preparative that would include novel approaches to protein purification as well as improvements in cell and organ culture. The actual techniques are equally inclusive ranging from aptamers to zymology. The journal has been particularly active in: -Analytical techniques for biological molecules- Aptamer selection and utilization- Biosensors- Chromatography- Cloning, sequencing and mutagenesis- Electrochemical methods- Electrophoresis- Enzyme characterization methods- Immunological approaches- Mass spectrometry of proteins and nucleic acids- Metabolomics- Nano level techniques- Optical spectroscopy in all its forms. The journal is reluctant to include most drug and strictly clinical studies as there are more suitable publication platforms for these types of papers.