Pipette-Tip-Enabled Digital Nucleic Acid Analyzer for COVID-19 Testing with Isothermal Amplification

Herein, a pipette-tip-enabled digital nucleic acid analyzer for high-performance COVID-19 testing is demonstrated. This is achieved by digital loop-mediated isothermal amplification (digital LAMP or dLAMP) using common laboratory equipment and materials. It is shown that simply fixing a glass capillary inside conventional pipette tips enables the generation of monodisperse, water-in-oil microdroplets with benchtop centrifugation. It is shown that using LAMP, the ORF1a/b gene, a standard test region for COVID-19 screening, can be amplified without a thermal cycler. The amplification allows counting of fluorescent microdroplets so that Poisson analysis can be performed to allow quantification with a limit of detection that is 1 order of magnitude better than those of nondigital techniques and comparable to those of commercial dLAMP platforms. It is envisioned that this work will inspire studies on ultrasensitive digital nucleic acid analyzers demanding both sensitivity and accessibility, which is pivotal to their large-scale applications.

Automated summary

A pipette-tip-enabled digital nucleic acid analyzer for high-performance COVID-19 testing is demonstrated in this study. By using digital LAMP technology, the ORF1a/b gene can be amplified without the need for a thermal cycler, allowing for the generation of monodisperse microdroplets with benchtop centrifugation. The method offers a significant improvement in sensitivity over nondigital techniques and compares favorably to commercial dLAMP platforms, showing potential for large-scale applications of ultrasensitive digital nucleic acid analyzers.