Profiling RT-LAMP tolerance of sequence variation for SARS-CoV-2 RNA detection

Abstract

The ongoing SARS-CoV-2 pandemic has necessitated a dramatic increase in our ability to conduct molecular diagnostic tests, as accurate detection of the virus is critical in preventing its spread. However, SARS-CoV-2 variants continue to emerge, with each new variant potentially affecting widely-used nucleic acid amplification diagnostic tests. RT-LAMP has been adopted as a quick, inexpensive diagnostic alternative to RT-qPCR, but as a newer method, has not been studied as thoroughly. Here we interrogate the effect of SARS-CoV-2 sequence mutations on RT-LAMP amplification, creating 523 single point mutation "variants" covering every position of the LAMP primers in 3 SARS-CoV-2 assays and analyzing their effects with over 4,500 RT-LAMP reactions. Remarkably, we observed only minimal effects on amplification speed and no effect on detection sensitivity at positions equivalent to those that significantly impact RT-qPCR assays. We also created primer sets targeting a specific short deletion and observed that LAMP is able to amplify even with a primer containing multiple consecutive mismatched bases, albeit with reduced speed and sensitivity. This highlights RT-LAMP as a robust technique for viral RNA detection that can tolerate most mutations in the primer regions. Additionally, where variant discrimination is desired, we describe the use of molecular beacons to sensitively distinguish and identify variant RNA sequences carrying short deletions. Together these data add to the growing body of knowledge on the utility of RT-LAMP and increase its potential to further our ability to conduct molecular diagnostic tests outside of the traditional clinical laboratory environment.

Fig 1. Mutation position effects on RT-LAMP amplification.

Plots of the effects on amplification speed relative to the WT primer set for all 3 assays explored, As1e (blue circle), E1 (red square), N2 (green triangle). (A) F3 primer, (B) B3 primer, (C) FIP primer, (D) BIP primer, (E) Loop F primer, (F) Loop B primer.

Fig 2. Detection of target RNAs by SGFwt and SGFdel molecular beacons.

(A) Sequence comparison for wt, SGF deletion, SGFwt-MB and SGFdel-MB. Dashes: bases deleted in SGF deletion; Bold: LNA base; Underlined: stem region; Italics: non-target sequence, attached fluorophores and quenchers. LAMP reactions with either WT RNA (left panels) or B.1.1.7 RNA (right panels) in the presence of (B) SYTO-9, (C) SGFdel-MB, or (D) SGFwt-MB. The primer set amplifies both the wt and B.1.1.7 RNA with similar efficiency as detected with SYTO-9 (B). When beacon was added as a reporter, both SGFdel-MB (C) and SGFwt-MB (D) detected only their intended template RNAs from 50–10,000 copies.