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 multitarget 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 multitarget redundancy. When challenged with extracted human samples the multiplex assay showed 87% or better sensitivity (of 30 positive samples), with 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction (of 21 positive samples), and 100% specificity (of 60 negative samples). 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. IMPORTANCE The COVID-19 pandemic has had tremendous impact, and the ability to perform molecular diagnostics in resource limited settings has emerged as a key resource for mitigating spread of the disease. One challenge in COVID-19 diagnosis, as well as other viruses, is ongoing mutation that can allow viruses to evade detection by diagnostic tests. We developed a test that detects multiple parts of the virus genome in a single test to reduce the chance of missing a virus due to mutation, and it is designed to be simpler and faster than typical laboratory tests while maintaining high sensitivity. This capability is enabled by a novel fluorescent probe technology that works with a simple constant temperature reaction condition.

Keywords: coronavirus; diagnostics; molecular methods.

FIG 1

Multiplex RT-LAMP (mRT-LAMP) fluorescence detection by Universal Displacement Probes (UDP). (A) UDP incorporation during LAMP amplification and activation by displacement of quenching strand. Primer and probe refer to loop (L), adapter (A), and quencher (Q), with complementary sequences denoted with the suffix “c” (e.g., “Lc” is the reverse complement “L”). (B) Two-channel fluorescence detection of multiplexed redundant LAMP products (6-FAM) and shared-primer IAC (TEX615) by UDPs. Primer designations refer to forward (F), backward (B), and loop (L) using conventional LAMP terminology.

FIG 2

Analytical performance of mRT-LAMP for SARS-CoV-2. (A) Characteristic amplification of multiplexed SARS CoV-2 targets and internal amplification control (IAC) with real-time fluorescence detection by universal displacement probes (UDP). Single representative run with 200 copies of synthetic RNA input or a no template control (NTC). (B) Analytical sensitivity of multiplexed SARS-CoV-2 target and IAC. IAC amplifications (bottom) correspond to target amplifications (top). Target synthetic RNA input: 2,000 (n = 3), 200 (n = 3), 20 (n = 3), 10 (n = 3), or 5 copies per reaction (n = 4); and NTC (n = 3). (C) Time to detect signals from SARS-CoV-2 and IAC for reactions from panel B.

FIG 3

mRT-LAMP amplification of extracted nasal specimens. Samples confirmed as Negative or Positive for SARS-CoV-2 by RT-PCR panel (N1, N2, RP) were amplified by duplicate mRT-LAMP reactions. mRT-LAMP signals for SARS-CoV-2 are shown in blue, and IAC signals are shown in orange with detected Threshold time (Tt) or “Not Detected” reported for both in all reactions; replicate pairs for each signal are connected by a line segment. Mean copy number was derived from qPCR results of N1, N2 PCR (see Table S3).