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 coronavirus disease 2019 (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.

Two Years into the COVID-19 Pandemic: Lessons Learned

“… 376 , 378 Since the emergence of SARS-CoV-2, many RT-LAMP assays have been developed for the diagnosis of this virus in several types of clinical samples, including saliva, serum, nasopharyngeal swabs, oropharyngeal swabs, and urine. 370 , 379 − 386 In general, RT-LAMP assays have been designed for different targets in the SARS-COV-2 genome (ORF1ab, N protein, E protein, RdRP, and M protein), and the clinical performances of many RT-LAMP assays have been compared with that of RT-qPCR. 315 , 385 , 387 Efforts to decrease the cost and simplify the RT-LAMP workflow for testing patient samples are in progress using protocols without RNA extraction from patient specimens and in-house-produced enzymes, 388 − 390 with the possibility to scale up COVID-19 diagnostics.…”

“…Considering its advantages of high specificity and sensitivity, rapid amplification, simple operation, and low cost, RT-LAMP has potential applications for the diagnosis of many infectious diseases. , Since the emergence of SARS-CoV-2, many RT-LAMP assays have been developed for the diagnosis of this virus in several types of clinical samples, including saliva, serum, nasopharyngeal swabs, oropharyngeal swabs, and urine. ,− In general, RT-LAMP assays have been designed for different targets in the SARS-COV-2 genome (ORF1ab, N protein, E protein, RdRP, and M protein), and the clinical performances of many RT-LAMP assays have been compared with that of RT-qPCR. ,, Efforts to decrease the cost and simplify the RT-LAMP workflow for testing patient samples are in progress using protocols without RNA extraction from patient specimens and in-house-produced enzymes, − with the possibility to scale up COVID-19 diagnostics. With regard to the limit of detection (LoD), the majority of RT-LAMP assays should have LoDs ranging from 200 to 100 copies per reaction, , while a few have demonstrated LoDs as low as 10 copies per reaction or even 1 copy per reaction. − Currently, there are 14 diagnostic devices based on RT-LAMP assays that have been granted Emergency Use Authorization (EUA) by the FDA ().…”

Development and Validation of Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) as a Simple and Rapid Diagnostic Tool for SARS-CoV-2 Detection

“…Numerous studies on RNA clinical samples with a high Cq value (>30), in which the results did not pick up by the RT-LAMP assay or took a longer time > 35 min to be detected, which could affect some negative samples and might cause false-positive results [ 32 , 33 , 34 ]. Other studies found the cutoff of RT-LAMP when the Cq value was 32 or more [ 35 , 36 ]. Therefore, further optimization is needed to identify the Cq breakpoint for this assay and to improve it by increasing the input amount of RNA in the reaction.…”

Development of loop-mediated isothermal amplification (LAMP) assays using five primers reduces the false-positive rate in COVID-19 diagnosis

“…The improvement was evident with final concentrations between 40 and 60 mM in a 25 µL reaction volume, with 40 mM being the optimum recommended concentration https://international.neb.com 24 . Most studies reported this enhancement from GuHCl on RNA-extracted clinical samples but not in direct ones 22 , 23 . Based on that, we used 40 mM GuHCl in the colorimetric and fluorometric RT-LAMP reactions; both resulting in 3–7-min earlier amplifications in positive reactions with GuHCl.…”