Division of Chemistry and Chemical Engineering, California Institute of Technology 1200 E. California Boulevard, Pasadena, CA 91125, USA.
Division of Biology and Biological Engineering, California Institute of Technology 1200 E. California Boulevard, Pasadena, CA 91125, USA.
Nucleic Acids Res. 2020 Apr 17;48(7):e42. doi: 10.1093/nar/gkaa099.
Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.
等温扩增检测法，如环介导等温扩增（LAMP），在传染病快速诊断的发展方面具有很大的应用潜力，因为其具有高灵敏度、病原体特异性和在床边实施的潜力。然而，消除非特异性扩增仍然是优化 LAMP 检测法的一个关键挑战。在这里，我们使用衣原体 DNA 作为临床相关的靶标，并采用高通量测序作为分析工具，研究非特异性扩增的潜在机制。然后，我们开发了一种具有高分辨率熔解温度（HRM）分析的实时数字 LAMP（dLAMP），并使用这种单分子方法分析了大约 120 万个扩增事件。我们表明，单分子 HRM 提供了有关 LAMP 中特异性和非特异性扩增的见解，这些见解很难从批量测量中推断出来。我们使用具有 HRM 的实时 dLAMP 来评估聚合酶酶之间的差异、检测参数（例如时间、速率或荧光强度）的影响，以及背景人 DNA 的影响。通过区分真阳性和假阳性，HRM 可以确定最佳检测和分析参数，从而在数字等温扩增检测中实现最低检测限（LOD）。
