Padlock probe-based rolling circle amplification lateral flow assay for point-of-need nucleic acid detection.
Padlock probe-based rolling circle amplification lateral flow assay for point-of-need nucleic acid detection.
复制标题
DOI：
10.1039/d1an00399b
复制
发表时间：
2021-06-28
期刊：
影响因子：
--
通讯作者：
Henry CS
中科院分区：
文献类型：
--
作者：
Jain S;Dandy DS;Geiss BJ;Henry CS
敏感、可靠和具有成本效益的病原体检测在临床诊断和治疗、水和食品安全、环境监测、生物安全和流行病学方面有着广泛的应用。核酸扩增试验（NAATs），如PCR和等温扩增方法提供了出色的分析性能，并且比传统的基于培养的方法的周转时间要快得多。然而，NAATs的固有成本和复杂性限制了它们在资源有限的环境和发展中国家的应用。为了帮助解决这一迫切需求，我们开发了一种基于挂锁探针滚动环扩增（PLRCA）、核酸酶保护（NP）和侧流检测（LFA）的灵敏核酸分析方法，称为PLAN-LFA，可用于资源有限的环境。该检测包括将挂锁探针与靶标进行液相杂交，将探针序列特异性连接形成圆形模板，在聚合酶和标记探针DNA的存在下进行等温滚圈扩增。RCA产物是一个长，线性连接的单链DNA，包含标记探针的结合位点。然后将样品暴露于选择性切割单链DNA的核酸酶中，双链标记探针被保护免受核酸酶消化，并以侧流免疫分析格式检测，以提供结果的视觉比色读出。我们利用PLAN-LFA平台开发了针对β -内酰胺酶耐药基因的特异性检测方法，用于监测抗菌素耐药性和2019年发现的新型冠状病毒-2 （SARS-CoV-2）。该检测提供1.1 pM靶DNA（或1.3×106拷贝/反应）的检测限。我们还通过对DNA和RNA靶点进行分析，并在没有任何样品预处理步骤的情况下对唾液、植物组织提取物和细菌培养等复杂样品基质进行分析，证明了该方法的通用性和鲁棒性。
Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs) such as PCR and isothermal amplification methods provide excellent analytical performance and significantly faster turnaround times than conventional culture-based methods. However, the inherent cost and complexity of NAATs limit their application in resource-limited settings and the developing world. To help address this urgent need, we have developed a sensitive method for nucleic acid analysis based on padlock probe rolling circle amplification (PLRCA), nuclease protection (NP) and lateral flow detection (LFA), referred to as PLAN-LFA, that can be used in resource-limited settings. The assay involves solution-phase hybridization of a padlock probe to target, sequence-specific ligation of the probe to form a circular template that undergoes isothermal rolling circle amplification in the presence of a polymerase and a labeled probe DNA. The RCA product is a long, linear concatenated single-stranded DNA that contains binding sites for the labeled probe. The sample is then exposed to a nuclease which selectively cleaves single-stranded DNA, the double-stranded labeled probe is protected from nuclease digestion and detected in a lateral flow immunoassay format to provide a visual, colorimetric readout of results. We have developed specific assays targeting beta-lactamase resistance gene for monitoring of antimicrobial resistance and Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2, the novel coronavirus discovered in 2019) using the PLAN-LFA platform. The assay provides a limit of detection of 1.1 pM target DNA (or 1.3×106 copies/reaction). We also demonstrate the versatility and robustness of the method by performing analysis on DNA and RNA targets, and perform analysis in complex sample matrices like saliva, plant tissue extract and bacterial culture without any sample pretreatment steps.
登录
查看更多内容
影响因子：
6.4
作者：
Koczula KM;Gallotta A
通讯作者：
Gallotta A
基于挂锁探针的指数滚环扩增对 MicroRNA 进行高特异性和超灵敏等温检测
影响因子：
7.4
作者：
Liu, Haiyun;Li, Lu;Tang, Bo
通讯作者：
Tang, Bo
影响因子：
2.9
作者：
Kaocharoen, Sirada;Wang, Bin;Meyer, Wieland
通讯作者：
Meyer, Wieland
影响因子：
4.5
作者：
Lagunavicius, Arunas;Merkiene, Egle;Janulaitis, Arvydas
通讯作者：
Janulaitis, Arvydas
影响因子：
48
作者：
Larsson, C;Koch, J;Nilsson, M
通讯作者：
Nilsson, M