Abstract
Abstract Background Loop Mediated Isothermal Amplification (LAMP) has become a widely used method for detection of target nucleic acids (DNA and RNA) as it offers a robust and simple alternative to PCR. LAMP is particularly well suited to point of care (POC) applications such as COVID-19 diagnostics because only a single incubation temperature is required for nucleic acid amplification and the technology is compatible with simple detection strategies, including colorimetric or lateral flow readouts. To further increase the utility of LAMP in POC molecular diagnostic workflows, elimination of cold chain requirements for reagent shipment and storage is desired. In this study, we investigate single and multiplex detection of several viral infectious diseases using lyophilized RT-LAMP reagents. Methods Assays targeting SARS-CoV-2, FluA, FluB and a host control nucleic acid were evaluated using various detection methods with lyophilized RT-LAMP reagents. Compatibility was established with endpoint LAMP detection strategies, such as colorimetric dyes, and real time multiplex LAMP detection, which is increasingly finding utility in POC testing for common respiratory infections that present with similar symptoms. Results Assays employing lyophilized RT-LAMP reagents maintained robust detection of viral respiratory targets within 30 min at 65°C using various detection strategies. Colorimetric dyes such as eriochrome back T and calcein resulted in successful visual readout upon amplification while, one prominent colorimetric dye, hydroxy naphthol blue, was found to be incompatible with the lyophilized reagents used in the study. Additionally, simple hybridization probes permitted simultaneous detection of SARS-CoV-2, FluA and FluB in a single reaction. Conclusion LAMP is a robust, simple, isothermal amplification strategy that continues to enable testing beyond traditional laboratory settings. Lyophilized reagents extend the utility of LAMP in POC settings regardless of the detection strategy and will be important factors in the extension of this powerful molecular diagnostic tool to decentralized, field, and at-home testing applications.
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