Key Laboratory of Clinical Laboratory Diagnostics (Chinese Ministry of Education), College of Laboratory Medicine, Chongqing Medical Laboratory Microfluidics and SPRi Engineering Research Center, Chongqing Medical University, Chongqing 400016, China.
Department of Laboratory Medicine, Luzhou Traditional Chinese Medicine Hospital, Luzhou 646000, China.
Anal Chem. 2023 Aug 22;95(33):12487-12496. doi: 10.1021/acs.analchem.3c02232. Epub 2023 Aug 3.
Loop-mediated isothermal amplification (LAMP) is a commonly used alternative to PCR for point-of-care detection of nucleic acids due to its rapidity, sensitivity, specificity, and simpler instrumentation. While dual-labeled TaqMan probes are widely used in PCR for single-nucleotide polymorphism (SNP) genotyping, real-time LAMP primarily relies on turbidimetry or intercalator fluorescence measurements, which can be non-specific and generate false-positive results. In this study, we propose a closed-tube, dual-labeled RNA-modified probes and RNase H II-assisted real-time LAMP (RART-LAMP) method for SNP genotyping. Our findings indicate that (1) fluorescence signals were predominantly derived from probe hydrolysis rather than hybridization, (2) temperature-controlled hybridization between the probe and template ensured the specificity of SNP analysis, and (3) RNase H II hydrolysis between the target containing SNP sites and probes did not exhibit sequence specificity. Our RART-LAMP approach demonstrated excellent performance in genotyping C677T clinical samples, including gDNA extracted from blood, saliva, and swabs. More importantly, saliva and swab samples could be directly analyzed without any pretreatment, indicating promising prospects for nucleic acid analysis at the point of care in resource-limited settings.
环介导等温扩增（LAMP）由于其快速、灵敏、特异和仪器简单等特点，是一种常用于即时检测核酸的替代聚合酶链反应（PCR）的方法。虽然双标记 TaqMan 探针广泛应用于 PCR 中的单核苷酸多态性（SNP）基因分型，但实时 LAMP 主要依赖浊度或嵌入荧光测量，这可能是非特异性的，并产生假阳性结果。在本研究中，我们提出了一种封闭管、双标记 RNA 修饰探针和 RNase H II 辅助实时 LAMP（RART-LAMP）方法用于 SNP 基因分型。我们的研究结果表明：（1）荧光信号主要来自探针水解而不是杂交；（2）探针与模板之间的温度控制杂交确保了 SNP 分析的特异性；（3）靶标中含有 SNP 位点的探针之间的 RNase H II 水解没有序列特异性。我们的 RART-LAMP 方法在 C677T 临床样本的基因分型中表现出优异的性能，包括从血液、唾液和拭子中提取的 gDNA。更重要的是，唾液和拭子样本可以直接进行分析，无需任何预处理，这表明在资源有限的环境中，即时核酸分析具有广阔的前景。
