1
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
Pedro A. Alves
1,2*
, Ellen G. de Oliveira
1
, Ana Paula M. Franco-Luiz
1
, Letícia T. Almeida
1
;
Amanda B. Gonçalves
1
, Iara A. Borges
1
, Flávia de S. Rocha
1
; Raissa P. Rocha
2
, Matheus F.
Bezerra
3
; Pâmella Miranda
4
; Flávio D. Capanema
5
; Henrique R. Martins
6,7
; Gerald Weber
4
;
Santuza M. R. Teixeira
2
, Gabriel Luz Wallau
8
; Rubens L. do Monte-Neto
1*
1
Instituto René Rachou – Fundação Oswaldo Cruz, Belo Horizonte, 30190-009, Minas Gerais,
Brasil;
2
Centro de Tecnologia em Vacinas, Belo Horizonte, 31310-260, Minas Gerais, Brasil
3
Depertamento de Microbiologia, Instituto Aggeu Magalhães – Fundação Oswaldo Cruz, Recife,
50670-420, Pernambuco, Brasil
4
Departamento de Física, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901,
Minas Gerais, Brasil
5
Núcleo de Inovação Tecnológica – Fundação Hospitalar do Estado de Minas Gerais, Belo
Horizonte, 30150-260, Minas Gerais, Brasil
6
Visuri Equipamentos e Serviços, Belo Horizonte, Minas Gerais, 31330-070, Minas Gerais Brasil
7
Departamento de Engenharia Elétrica – Universidade Federal de Minas Gerais, Belo Horizonte,
31270-901, Minas Gerais, Brasil
8
Departamento de Entomologia e Núcleo de Bioinformática, Instituto Aggeu Magalhães –
Fundação Oswaldo Cruz, Recife, 50670-420, Pernambuco, Brasil
*Corresponding authors: rubens.monte@fiocruz.br; pedro.alves@fiocruz.br
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 ± 2.7 viral genomic copies/μL when using RNA
extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral
transport medium. Typical RT-LAMP reactions were performed at 65 ºC 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
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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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.
Keywords: COVID-19; RT-LAMP; SARS-CoV-2; molecular test; colorimetric; respiratory virus
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted June 2, 2021. ; https://doi.org/10.1101/2021.05.26.21257488doi: medRxiv preprint
3
INTRODUCTION
Emerging viral infections continue to pose a major threat to global public health. In the
past decades different viral emergencies have been reported including the severe acute respiratory
syndrome coronavirus (SARS-CoV), H1N1 influenza, Middle East respiratory syndrome
coronavirus (MERS-CoV), Ebola vírus, Zika virus and most recently the new coronavirus has been
described, which cause COVID-19 (1,2). COVID-19 has as etiologic agent the severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the Coronaviridae family,
Betacoronavirus genus (3,4). People with COVID-19 have a wide range of symptoms reported
such as fever, cough, anosmia, ageusia, headache, fatigue, muscle or body aches, sore throat,
shortness of breath or difficulty breathing. Some of these symptoms help spread the virus, however
human-to-human transmission from infected individuals with no or mild symptoms has been
extensively reported (5,6). This outbreak has spread rapidly, as of May 2021, there were over 165
million confirmed COVID-19 cases with over 3,4 million deaths recorded worldwide
(https://coronavirus.jhu.edu/). Isolation and quarantine of infected individuals is essential to viral
spread and community dissemination of airborne pathogens and requiring an accurate, fast,
affordable, readily available tests for massive population testing. In contrast do antibody detection,
which may take weeks after the onset of the infection. Detection of viral RNA is the best way to
confirm the acute infection phase, the most important phase for viral shedding, so that rationally
managed social distancing and lockdown can be implemented (1,7).
Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is considered the
gold standard method for SARS-CoV-2 RNA detection, mainly targeting combinations of viral
genome regions that codes for nucleocapsid protein (N), envelope protein (E), RNA-dependent
RNA polymerase (RdRp) and other targets on the open reading frame (ORF1ab) (8). Although
RT-qPCR assays have played an important role un the SARS-CoV-2 diagnosis; the technique has
limitations for massive population testing such as processing time, the requirement of sophisticated
equipment, infrastructure and highly trained personnel, as well as costly reagents with high
demand and shortages around the world. Thus, developing complementary, inexpensive point-of-
care (PoC) methods that are rapid, simple, allowing the use of alternative reagents for COVID-19
diagnosis test, is urgently needed. Methods gathering these features can make affordable massive
testing campaigns, including contact tracing strategies in highly dense countries, saving lives (9–
17). In this regard reverse transcription loop-mediated isothermal amplification (RT-LAMP) has
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted June 2, 2021. ; https://doi.org/10.1101/2021.05.26.21257488doi: medRxiv preprint
4
been shown to be an affordable technique applied to detect different pathogens (18,19). RT-LAMP
has been used during Ebola outbreak (20,21) and for tracking Zika virus (22) or Wolbachia (23)
in Brazilian mosquitoes. The method relies on specific DNA amplification at constant temperature
without the need for sophisticated thermal cyclers (24). The amplified products can be visually
detected by magnesium pyrophosphate precipitation; fluorescence emission by DNA intercalating
dyes; agarose gel electrophoresis; lateral flow immunochromatography; magnesium chelating
color indicators and pH-dependent colorimetric reaction that changes from fuchsia (pink) to yellow
(positive result) due to proton release during nucleic acid amplification (25) (Figure 1). The
possibility of accessing results by naked eye, made RT-LAMP an exciting alternative that
facilitates the use of COVID-19 molecular testing. Simple, scalable, cost-effective RT-LAMP-
based alternatives for SARS-CoV-2 detection, has emerged during pandemics including protocols
for viral inactivation, quick run, RNA extraction-free and LAMP-associated CRISPR/Cas
strategies (10,11,14,16,17,26–32). On April 14
th
, 2020, the RT-LAMP received the emergency use
authorization from the United States Food and Drug Administration for SARS-CoV-2 detection in
COVID-19 diagnostics.
In this study, we validate a colorimetric RT-LAMP assay to detect SARS-CoV-2 RNA in
clinical samples collected in different parts of Brazil, including samples with known SARS-CoV-
2 variants of interest and concern. After testing primers used by RT-LAMP SARS-CoV-2 RNA
detection targeting different regions, best results were achieved when using N gene or N/E genes-
based strategies. One hundred nasopharyngeal swabs collected in a guanidine-containing viral
transport medium (VTM) (33) from symptomatic hospitalized patients were tested. The clinical
validation revealed a sensitivity of 97% (87.4-99.4 95% CI) with samples ranging Ct values from
15 to 32 with 100% specificity. The use of RNA extraction-free samples was also tested, although
there is a loss in sensitivity. We also demonstrated that RT-LAMP is affordable for the detection
of more transmissible SARS-CoV-2 variants encompassing a number of genomic nucleotide
changes. Part of the results presented here are the research basis of OmniLAMP
®
SARS-CoV-2
kit which was approved by the Brazilian Heath Regulatory Agency for COVID-19 molecular
testing (Anvisa nº: 10009010368) as an alternative for massive decentralized diagnostic in Brazil,
that records the third-highest COVID-19 cases number worldwide (https://coronavirus.jhu.edu).
Together with vaccination, RT-LAMP for COVID-19 diagnosis could help to improve better life
quality during pandemics, offering an alternative molecular testing for monitoring lock-down
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted June 2, 2021. ; https://doi.org/10.1101/2021.05.26.21257488doi: medRxiv preprint
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measures; traveling restrictions; the return of universities, schools, kindergartens; sport leagues
(including the presence of audience, in the Olympics for example) activities with worldwide
impact.
Figure 1 – Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 RNA
detection and COVID-19 testing. Inactivated saliva samples or from nasopharyngeal swabs can processed for RNA
extraction previously or be directly added to RT-LAMP reaction. Colorimetric output can be achieved by different
sensors and can be read by naked eye. The whole procedure is rapid, simple and do not requires complex
infrastructures.
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted June 2, 2021. ; https://doi.org/10.1101/2021.05.26.21257488doi: medRxiv preprint
