Husseini Abbas Ali, Baydar Serap Yesilkir
Life Sciences and Biomedical Engineering Application and Research Centre, Istanbul Gelisim University, Istanbul, Türkiye.
Vocational School of health services, Istanbul Gelisim University, 34310, Istanbul, Türkiye.
Mol Biol Rep. 2023 Oct;50(10):8329-8336. doi: 10.1007/s11033-023-08730-9. Epub 2023 Aug 17.
The utilization of direct amplification of nucleic acid from lysate has attracted interest in the advancement of straightforward and economical point-of-care assays. Consequently, this study primarily focuses on the development of a rapid, precise, and cost-effective lateral flow biosensor for the convenient detection of HBV nucleic acid at the point-of-care. Furthermore, the study evaluates the effectiveness of the direct amplification method in comparison to purified nucleic acid samples within the context of LAMP-LF biosensing approaches.
The experiments conducted in this study utilized clinical serum samples that were confirmed as HBV-positive through real-time PCR assays. Sample preparation involved employing spin column nucleic acid purification and serum heat treatment. To amplify a 250 bp fragment of the HBV polymerase gene, three pairs of specific LAMP primers were utilized, which were biotin-labeled and FITC-labeled for detection purposes. Various incubation temperatures (ranging from 64 to 68 °C) and durations (30 min, 45 min, and 1 h) were investigated to determine the optimal conditions for the LAMP assay. The results were subsequently assessed through fluorometric analysis, white turbidity measurements, and lateral flow assay. Milenia HybriDetect1 strips, designed for immediate use, were employed to visualize the LAMP amplicons. Furthermore, the performance of the lateral flow biosensor was evaluated using 10-fold serial dilutions of a secondary standard containing a viral load of 10 IU/ml.
The optimization of the LAMP reaction was achieved at a temperature of 67 °C, resulting in significant turbidity after a 30-minute incubation period. When the spin column purification method was employed, varying test bands were observed for templates ranging from 10 IU/ml to 10 IU/ml viral load. However, when serum samples underwent heat treatment and the resulting supernatant was directly used for LAMP, the lateral flow assay was capable of detecting a minimum viral load of 10 IU/ml.
In resource-limited settings, the LAMP-LF assay presents a promising solution for HBV testing. However, it is important to note that direct amplification without DNA purification may diminish the performance of the approach.
直接从裂解物中扩增核酸的应用在简便且经济的即时检测方法的发展中引起了关注。因此，本研究主要致力于开发一种快速、精确且经济高效的侧向流动生物传感器，以便在即时检测点方便地检测乙肝病毒核酸。此外，本研究在环介导等温扩增-侧向流动生物传感方法的背景下，评估了直接扩增方法与纯化核酸样本相比的有效性。
本研究中进行的实验使用了通过实时聚合酶链反应检测确认为乙肝病毒阳性的临床血清样本。样品制备包括采用离心柱核酸纯化和血清热处理。为了扩增乙肝病毒聚合酶基因的250bp片段，使用了三对特异性环介导等温扩增引物，这些引物为生物素标记和异硫氰酸荧光素标记用于检测目的。研究了各种孵育温度（64至68°C）和持续时间（30分钟、45分钟和1小时），以确定环介导等温扩增测定的最佳条件。随后通过荧光分析、白色浊度测量和侧向流动测定来评估结果。设计用于即时使用的Milenia HybriDetect1试纸条用于可视化环介导等温扩增扩增子。此外，使用含有10IU/ml病毒载量的二级标准品的10倍系列稀释液评估侧向流动生物传感器的性能。
环介导等温扩增反应在67°C温度下实现了优化，孵育30分钟后产生明显的浊度。当采用离心柱纯化方法时，对于病毒载量从10IU/ml到10IU/ml的模板观察到不同的测试条带。然而，当血清样本经过热处理且所得上清液直接用于环介导等温扩增时，侧向流动测定能够检测到最低10IU/ml的病毒载量。
在资源有限的环境中，环介导等温扩增-侧向流动测定法为乙肝病毒检测提供了一种有前景的解决方案。然而，需要注意的是，未经DNA纯化的直接扩增可能会降低该方法的性能。
