用于从全血中分离核酸的简单电装置，优化用于即时检测脑损伤。

Simple Electric Device to Isolate Nucleic Acids from Whole Blood Optimized for Point of Care Testing of Brain Damage.

机构信息

Department of Physiology, School of Medicine, Kyungpook National University, Daegu 41944, Korea.

Clinical Omics Institute, Kyungpook National University, Daegu 41405, Korea.

出版信息

Curr Neurovasc Res. 2022;19(3):333-343. doi: 10.2174/1567202619666220903105805.

DOI:10.2174/1567202619666220903105805

PMID:36056832

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10009893/

Abstract

BACKGROUND

Detection or monitoring of brain damage is a clinically crucial issue. Nucleic acids in the whole blood can be used as biomarkers for brain injury. Polymerase chain reaction (PCR) which is one of the most commonly used molecular diagnostic assays requires isolated nucleic acids to initiate amplification. Currently used nucleic acid isolation procedures are complicated and require laboratory equipments.

OBJECTIVE

In this study, we tried to develop a simple and convenient method to isolate nucleic acids from the whole blood sample using a tiny battery-powered electric device. The quality of the isolated nucleic acids should be suitable for PCR assay without extra preparation.

METHODS

A plastic device with separation chamber was designed and printed with a 3D printer. Two platinum electrodes were placed on both sides and a battery was used to supply the electricity. To choose the optimal nucleic acid isolation condition, diverse lysis buffers and separation buffers were evaluated, and the duration and voltage of the electricity were tested. Western blot analysis and PCR assay were used to determine the quality of the separated nucleic acids.

RESULTS

2ul of whole blood was applied to the cathode side of the separation chamber containing 78 ul of normal saline. When the electricity at 5 V was applied for 5 min, nucleic acids were separated from segment 1 to 3 of the separation chamber. The concentration of nucleic acids peaked around 7~8 mm from cathode side. PCR assay using the separation buffer as the template was performed successfully both in conventional and realtime PCR methods. The hemoglobin in the whole blood did not show the inhibitory effect in our separation system and it may be due to structural modification of hemoglobin during electric separation.

CONCLUSION

Our simple electric device can separate nucleic acids from the whole blood sample by applying electricity at 5 V for 5 min. The separation buffer solution taken from the device can be used for PCR assay successfully.

摘要

背景

脑部损伤的检测或监测是一个临床关键问题。全血中的核酸可用作脑损伤的生物标志物。聚合酶链反应（PCR）是最常用的分子诊断检测方法之一，它需要分离的核酸来启动扩增。目前使用的核酸分离程序复杂，需要实验室设备。

目的

在这项研究中，我们尝试开发一种简单方便的方法，使用微型电池供电的电动设备从全血样本中分离核酸。分离得到的核酸无需额外准备，其质量应适合 PCR 检测。

方法

设计并使用 3D 打印机打印了一个带有分离腔的塑料设备。在设备的两侧放置两个铂电极，并使用电池供电。为了选择最佳的核酸分离条件，我们评估了多种裂解缓冲液和分离缓冲液，并测试了电流的持续时间和电压。使用 Western blot 分析和 PCR 检测来确定分离核酸的质量。

结果

将 2ul 的全血应用于分离腔的阴极侧，分离腔中含有 78ul 的生理盐水。当施加 5V 的电流 5 分钟时，核酸从分离腔的第 1 到第 3 段中分离出来。核酸的浓度在离阴极侧 7-8mm 处达到峰值。使用分离缓冲液作为模板的 PCR 检测在常规和实时 PCR 方法中均成功进行。全血中的血红蛋白在我们的分离系统中没有表现出抑制作用，这可能是由于血红蛋白在电分离过程中发生了结构修饰。