DataSheet_1_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.pdf

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 × 109 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.

环介导等温扩增技术（Loop-mediated isothermal amplification，简称LAMP）作为一种稳健快速的诊断工具，被广泛应用于防止传染病的传播。然而，源自先前测试的LAMP扩增产物中的交叉污染问题，在基于LAMP的生物分析检测中一直存在。在本研究中，我们开发了一种针对携带污染消除和SARS-CoV-2在即时检测（Point-of-care，简称POC）中快速检测的环化尿嘧啶-DNA糖基化酶实时逆转录酶LAMP检测方法（Cod-uracil-DNA-glycosylase real-time reverse transcriptase LAMP assay，简称Cod-UNG-rRT-LAMP）。利用Cod-UNG-rRT-LAMP检测方法，SARS-CoV-2病毒可在扩增后的45分钟内检测到，最低可达2拷贝/µl（8拷贝/反应），同时每反应可消除2.63 ± 0.17 pg（相当于2.296 × 10^9拷贝）的污染物。使用Cod-UNG-rRT-LAMP检测方法对临床SARS-CoV-2样本进行分析，与实时逆转录聚合酶链反应（rRT-PCR）相比，显示出优异的一致性，相对准确率为98.2%，灵敏度为97.1%，特异性为95.2%。本研究获得的结果明确展示了Cod-UNG-rRT-LAMP检测方法在POC诊断SARS-CoV-2以及现场检测其他病原体中的应用可行性。