Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes

Abstract

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multi-target redundancy and an internal amplification control. A convenient and cost-effective target specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-COV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multi-target redundancy. When challenged with extracted human samples the multiplexed assay showed 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity. These results are further evidence that conventional laboratory methodologies can be leveraged at the point-of-care for robust performance and diagnostic stability over time.

Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection

“…Therefore, primer design decisions were based on performance-driven parameters such as computational estimates of primer properties (e.g., homodimers, hairpin structures, Tm, and %GC). Our recent in silico analysis revealed that our primers still have high coverage across new variants (8). The specific overlap with the U.S. Centers for Disease Control and Prevention (CDC) primers (Fig.…”

“…Harmony also uses novel universal probes containing entirely engineered sequences (8). The 5′ FAM or 5′ TEX 615 signal probes each have a 5′ sequence region hybridized to the complementary sequence region of its quencher probe that has 3′ Iowa Black FQ or 3′ BHQ-2, respectively.…”

RT-PCR/MALDI-TOF diagnostic target performance reflects circulating SARS-CoV-2 variant diversity in New York City

“…A number of recent reports have focused on variant panels designed to target a finite number of emergent mutations to discern variant identity, particularly in the first months of 2021. Most panels utilize RT-PCR or novel isothermal technologies (e.g., RT-LAMP, CRISPR/Cas-based, massspectrometry) to target specific polymorphisms in the S gene that have been ascribed to each of these variants 25,27,45,[48][49][50] . However, a recent deep analysis of circulating variants across the globe revealed that most of these signature mutations are not under positive selection 1 .…”

SARS-CoV-2 Detection Methods

“…The primers usually target regions in the N, S, E or ORF1ab gene; however, only one target can be used in each assay which limits its accuracy and increases the probability of false negative occurrence [38]. A multiplex RT-LAMP assay has also been developed which allows for more accurate detection since it targets two SARS-CoV-2 regions [39]. There are also tests that employ both CRISPR-Cas and RT-LAMP technology at the same time in order to circumvent some of their individual limitations [40,41].…”