Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application

Abstract

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the COVID-19 pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human β actin, and tested clinical samples in multiple countries. TTTT linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values ≤ 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 > Ct ≤ 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.

Development of Loop-mediated Isothermal Amplification (LAMP) Assays Using Five Primers Reduces the False-positive Rate in COVID-19 Diagnosis

“…The effect of guanidine hydrochloride (GuHCl) on the performance of E-ID1 primer set in both colorimetric and fluorometric RT-LAMP reactions was tested since it was reported to enhance the detection speed and efficiency (Dudley et al, 2020, Lu et al, 2022, Zhang, 2020b). Figure 3 (a) shows two sets of colorimetric RT-LAMP with and without the addition of GuHCl (40 mM) on positive clinical specimens, in addition to PC and NTC.…”

“…The improvement was evident with final concentrations between 40-60 mM in a 25 µL reaction volume, with 40 mM being the optimum recommended concentration <https://international.neb.com>\(Zhang et al, 2020b). Most studies reported this enhancement from GuHCl on RNA-extracted clinical samples but not in direct ones (Dudley et al, 2020, Lu et al, 2022). Based on that, we used 40 mM GuHCl in the colorimetric and fluorometric RT-…”

“…To address this issue, we hypothesize that a lower number of LAMP primers will reduce the false positivity rate, which is the most common limitation of the LAMP technique (Meagher et al, 2018, Odiwuor et al, 2022). The slower amplification rate can be improved by optimizing the performance of the developed colorimetric and fluorometric assays via increasing the enzyme’s concentration or adding primer binding enhancers like guanidine hydrochloride, as shown elsewhere (Dudley et al, 2020, Lu et al, 2022, Zhang, 2020b). In this work, we demonstrate the efficiency of using five primers to reduce misamplifications by comparing them with five primer sets targeting different genes such as RdRP, S , and N .…”

Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic control

“…(20,27,38–44). These publications describe many different primer sets and use patient samples without purification (“direct assays”) (some are summarized here: (42)) or with prior purification of RNA, many with optimized procedures where compelling data could be obtained that suggest suitable sensitivity of RT-LAMP for detection of infections in asymptomatic and symptomatic subjects. Yet, intrinsic limitations of simple RT-LAMP assays using colorimetric readouts without sequence-specific probes such as molecular beacons, complicate its practical implementation.…”

“…Since the onset of the pandemic, a large number of publications emerged describing the use and validation of RT-LAMP assays for SARS-CoV-2 diagnostics, i.e. (20,27,(38)(39)(40)(41)(42)(43)(44). These publications describe many different primer sets and use patient samples without purification ("direct assays") (some are summarized here: (42)) or with prior purification of RNA, many with optimized procedures where compelling data could be obtained that suggest suitable sensitivity of RT-LAMP for detection of infections in asymptomatic and symptomatic subjects.…”