Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2

Abstract

A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25-50 copies per reaction) to commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48 % (95 % CI 91.84%-99.96%) and NPA 100.00 % (95 % CI 93.84%-100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.

Keywords: Assay; Covid; Diagnostics; LAMP; Molecular; PCR; SARS-CoV-2; Validation.

Fig. 1

Map of the gene fragments from SARS-CoV-2 (Genbank ID MT2078.1) that were used for synthesizing the genetic construct template. Four fragments of specific SARS-CoV-2 regions (ORF1ab (nsp3,10–11), RdRP (nsp 12), and spike (S)) were concatenated into a single artificial construct 1097 base pairs long.

Fig. 2

Workflow used to analyze samples in this study. Images were obtained from the Centers for Disease Control (www.cdc.gov) and Bio-Rad Laboratories (www.bio-rad.com).