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LATE-PCR for LoC molecular diagnostics devices and its application to the sensitive detection of SARS-CoV-2
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1  Biomedical Research Foundation of the Academy of Athens, Athens 115 27, Greece (registering DOI)

The emergence of the novel coronavirus, SARS-CoV-2, has highlighted the need for rapid, accurate, and point-of-care diagnostic testing. Lab-on-a-Chip (LoC) devices offer the possibility to run such tests at a low cost while at the same time permitting the multiplexed detection of several viruses when coupled with microarray detection of the amplified products. Herein, we report the development of a protocol for the qualitative detection of SARS-CoV-2 through the design of appropriate primers that target evolutionary conserved regions of the virus. The proposed protocol relies on .an improved version of asymmetric RT-PCR, the Linear-After-The-Exponential (LATE)-PCR that uses primers that are deliberately designed for use at unequal concentrations. As a result, LATE-PCR exhibits similar efficiency to symmetric PCR while promoting accumulation of single-stranded products that can subsequently hybridize to a single strand DNA probe-spotted microarray. The performance of the developed LATE-PCR protocol was compared to that of symmetric RT-PCR and validated with the use of artificial viral RNA and nasopharyngeal swabs samples from real patients. Furthermore, and in order to illustrate its potential for integration into a biosensor platform, the amplicons were allowed to hybridize with probes covalently immobilized onto commercially-available functionalized glass, without the need of heat denaturation.

Keywords: SARS-CoV-2; asymmetric PCR; LATE PCR; microarray detection; molecular diagnostics