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实时逆转录环介导等温扩增中消除携带污染,用于即时检测点中 SARS-CoV-2 病毒的快速检测。

Elimination of Carryover Contamination in Real-Time Reverse Transcriptase Loop-Mediated Isothermal Amplification for Rapid Detection of the SARS-CoV-2 Virus in Point-of-Care Testing.

机构信息

Biolabchip Group, Department of Bioengineering, Technical University of Denmark, Lyngby, Denmark.

Laboratory of Applied Micro and Nanotechnology (LAMINATE), Department of Bioengineering, Technical University of Denmark, Lyngby, Denmark.

出版信息

Front Cell Infect Microbiol. 2022 Apr 20;12:856553. doi: 10.3389/fcimb.2022.856553. eCollection 2022.

Abstract

Loop-mediated isothermal amplification (LAMP) is being used as a robust rapid diagnostic tool to prevent the transmission of infectious diseases. However, carryover contamination of LAMP-amplified products originating from previous tests has been a problem in LAMP-based bio-analytical assays. In this study, we developed a Cod-uracil-DNA-glycosylase real-time reverse transcriptase LAMP assay (Cod-UNG-rRT-LAMP) for the elimination of carryover contamination and the rapid detection of SARS-CoV-2 in point-of-care (POC) testing. Using the Cod-UNG-rRT-LAMP assay, the SARS-CoV-2 virus could be detected as low as 2 copies/µl (8 copies/reaction) within 45 min of amplification and 2.63 ± 0.17 pg (equivalent to 2.296 × 10 copies) of contaminants per reaction could be eliminated. Analysis of clinical SARS-CoV-2 samples using the Cod-UNG-rRT-LAMP assay showed an excellent agreement with a relative accuracy of 98.2%, sensitivity of 97.1%, and specificity of 95.2% in comparison to rRT-PCR. The results obtained in this study clearly demonstrate the feasibility of the use of the Cod-UNG-rRT-LAMP assay for applications toward the POC diagnosis of SARS-CoV-2 and on-site testing of other pathogens.

摘要

环介导等温扩增 (LAMP) 被用作一种强大的快速诊断工具,以防止传染病的传播。然而,来自先前测试的 LAMP 扩增产物的交叉污染一直是基于 LAMP 的生物分析测定中的一个问题。在这项研究中,我们开发了一种 Cod-尿嘧啶-DNA-糖基化酶实时逆转录 LAMP 检测法(Cod-UNG-rRT-LAMP),用于消除交叉污染并在即时检测(POC)中快速检测 SARS-CoV-2。使用 Cod-UNG-rRT-LAMP 检测法,在扩增 45 分钟内可以检测到低至 2 拷贝/µl(8 拷贝/反应)的 SARS-CoV-2 病毒,并且可以消除每个反应中 2.63 ± 0.17 pg(相当于 2.296×10 拷贝)的污染物。使用 Cod-UNG-rRT-LAMP 检测法分析临床 SARS-CoV-2 样本,与 rRT-PCR 相比,具有 98.2%的相对准确性、97.1%的灵敏度和 95.2%的特异性。本研究的结果清楚地表明,Cod-UNG-rRT-LAMP 检测法可用于 SARS-CoV-2 的即时检测诊断和其他病原体的现场检测。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/540e/9065284/2c6c566a4455/fcimb-12-856553-g001.jpg

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