Simple and Highly Sensitive Molecular Diagnosis of Zika Virus by Lateral Flow Assays

We have developed a simple, user-friendly, and highly sensitive Zika virus (ZIKV) detection method by incorporating optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) and a lateral flow assay (LFA). The optimized RT-LAMP reaction was carried out using <i>Bst</i> 3.0 polymerase, which has robust and fast isothermal amplification performance even in the presence of high concentrations of inhibitors; this permitted the amplification of ZIKV RNA in pure water and human whole blood. In addition, the strong reverse transcription activity of <i>Bst</i> 3.0 polymerase enabled specific ZIKV RNA amplification without extra addition of reverse transcriptase. The RT-LAMP condition was optimized by adjusting the Mg²⁺ and dNTP mix concentration to extirpate nontarget amplification, which is caused by nonspecific primer dimers amplification. After 30 min of RT-LAMP reaction, the resultant amplicons were simply and rapidly analyzed by the LFA test in less than 5 min. The optimized RT-LAMP combined with the LFA allowed specific ZIKV RNA detection down to the single copy level within 35 min

Enhancing Loop-Mediated Isothermal Amplification through Nonpowered Nanoelectric Preconcentration

The global threat posed by the COVID-19 pandemic has catalyzed the development of point-of-care (POC) molecular diagnostics. While loop-mediated isothermal amplification (LAMP) stands out as a promising technique among FDA-approved methods, it is occasionally susceptible to a high risk of false positives due to nonspecific amplification of a primer dimer. In this work, we report an enhancing LAMP technique in terms of assay sensitivity and reliability through streamlined integration with a nonpowered nanoelectric preconcentration (NPP). The NPP, serving as a sample preparation tool, enriched the virus concentration in samples prior to the subsequent LAMP assay. This enrichment enabled not only to achieve more sensitive assay but also to shorten the assay time for all tested clinical samples by ∼10 min compared to the conventional LAMP. The shortened assay time suppresses the occurrence of nonspecific amplification by not providing the necessary incubation time, effectively suppressing misidentification by false positives. Utilizing this technique, we also developed a prototype of the POC NPP-LAMP kit. This kit offers a streamlined diagnostic process for nontrained individuals, from the sample enrichment, transfer of the enriched sample to LAMP assays, which facilitates on-site/on-demand diagnosis of SARS-CoV-2. This development holds the potential to contribute toward preventing not only the current outbreak but also future occurrences of pandemic viruses