Molecular Characterization of Increased Amplicon Lengths in SARS-CoV-2 Reverse Transcription Loop-Mediated Isothermal Amplification Assays.

ABSTRACT

Loop-mediated isothermal amplification (LAMP) is a power tool for the amplification of specific RNA and DNA targets. Much like PCR, LAMP requires primers that surround a target amplification region and generates exponential product through a unique highly specific daisy-chain single-temperature amplification reaction. However, until recently, attempts to amplify targets of greater than 200 base pairs (bp) have been mostly unsuccessful and limited to short amplicon targets of less than 150 bp. Although short amplicons have the benefit of a rapid detection (<40 min), they do not allow for the prediction of RNA integrity based on RNA length and possible intactness. In this study, 8 primer sets were developed using 2 LAMP primer-specific software packages against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid gene with insert lengths ranging from 262 to 945 bp in order to amplify and infer the integrity of viral RNA. Because these amplification lengths are greater than the current methods that use an insert length of 130 or less, they require a longer incubation, modified primer and temperature strategies, and the addition of specific adjuncts to prevent nonspecific amplification. This proof of concept study resulted in successful reverse transcription LAMP reactions for amplicon targets of 262, 687, 693, and 945 bp using a clinical nasopharyngeal NP sample, purified SARS-CoV-2 RNA, and crude lysate containing inactivated virus.