Clinical validation of colorimetric RT-LAMP, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to detect SARS-CoV-2 variants of concern

Abstract

The COVID-19 pandemics unfolded due to the widespread SARS-CoV-2 transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric RT-LAMP (reverse transcription loop-mediated isothermal amplification) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 viral genomic copies/uL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65 C for 30 min. When compared to RT-qPCR, up to Ct value 32, RT-LAMP presented 97% (87.4-99.4% 95% CI) sensitivity and 100% (86.2-100%) specificity for SARS-CoV-2 RNA detection targeting N gene. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI), such as variants occurring in Brazil named P.1, P.2, B.1.1.374 and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipments, infrastructure and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread

“…RT-LAMP reactions (12.5 µL total volume) contained 1x WarmStart ® Colorimetric LAMP Master Mix (New England Biolabs, Ipswich, MA, USA; #M1800L), a primer set composed of 1.6 µM FIP/BIP internal primers, 0.4 µM LF/LB loop primers and 0.2 µM F3/B3 external primers, and 1.25 µL of DGS:saliva mixture in 1:1 ratio, previously centrifuged at 1000× g for 30 s. Previously published primer sets targeting different regions of SARS-CoV-2 genome and the human gene ACTB were used (Table S1). All primer sets targeting SARS-CoV-2 have already been tested for specificity against other respiratory pathogens [5][6][7][8][9]. RT-LAMP reactions were carried out at 65 • C for 30 to 40 min.…”

Current trends in COVID-19 diagnosis and its new variants in physiological fluids: Surface antigens, antibodies, nucleic acids, and RNA sequencing

“…The amplified genome can be analyzed visually using metal-sensitive indicators, pH-sensitive dyes, gel electrophoresis, turbidity measurements after magnesium pyrophosphate-induced precipitation, and fluorescent dyes ( Fig. 11 ) [ 127 ].

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“…RT-LAMP is particularly advantageous for the detection of viral nucleic acids because it can be performed with the naked eye [ 127 ]. Of note, RT-LAMP has higher specificity and sensitivity, with no false-positive results reported [ 120 ].…”