Name: Forfatter Spotlight: Fremme patogendiagnostikk med standardisert LAMP
Uploaded: 2023-09-08T13:55:00
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP at JoVE.com

Dette arbeidet ble finansiert av Sistema General de Regalías fra Colombia, stipendnummer BPIN 2020000100092, og Universidad Icesi - Convocatoria Interna, stipendnummer CA0413119. MFVT ble også finansiert av assisterende professoratmidler fra Universidad de los Andes. Finansiørene deltok ikke i utforming, gjennomføring av aktiviteter, datainnsamling og dataanalyse og utarbeiding av manus. Vi takker Universitetssykehuset Fundación Valle del Lili for viralt RNA fra Sars-CoV-2-prøver og Dr. Alvaro Barrera-Ocampo for kommentarene til manuskriptet.

Prøvene som ble brukt ble levert av det kliniske laboratoriet ved Fundación Valle del Lili universitetssykehus og tilsvarte det rensede RNA fra pasienter som testet positivt for COVID-19 ved bruk av RT-qPCR-teknikken. Alle pasienter ga informert samtykke til forskning, og denne studien ble godkjent av den bioetiske komiteen for menneskelige studier fra Fundación Valle del Lili universitetssykehus.
1. RT-LAMP primer design og klargjøring
MERK: LAMP...

Bruke kolorimetrisk og RT-LAMP-analyse for å oppdage SARS-CoV-2 

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Who coronavirus (COVID-19) dashboard (no date). , (2023).</li><li>Ibrahim, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiologic+surveillance+for+controlling+Covid-19+pandemic:+types,+challenges+and+implications.">Epidemiologic surveillance for controlling Covid-19 pandemic: types, challenges and implications.</a> Journal of Infection and Public Health. 13 (11), 1630-1638 (2020).</li><li>Rojas-Gallardo, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COVID-19+in+Latin+America:+Contrasting+phylodynamic+inference+with+epidemiological+surveillance.+(Molecular+epidemiology+of+COVID-19+in+Latin+America).">COVID-19 in Latin America: Contrasting phylodynamic inference with epidemiological surveillance. (Molecular epidemiology of COVID-19 in Latin America).</a> medRxiv. , (2020).</li><li>Liu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+rate+of+RT-PCR+detection+of+SARS-CoV-2+infection+in+4880+cases+from+one+hospital+in+Wuhan,+China,+from+Jan+to+Feb+2020.">Positive rate of RT-PCR detection of SARS-CoV-2 infection in 4880 cases from one hospital in Wuhan, China, from Jan to Feb 2020.</a> Clinica Chimica Acta. 505, 172-175 (2020).</li><li>Kevadiya, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostics+for+SARS-CoV-2+infections.">Diagnostics for SARS-CoV-2 infections.</a> Nature Materials. 20 (5), 593-605 (2021).</li><li>Tomita, N., Mori, Y., Kanda, H., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP)+of+gene+sequences+and+simple+visual+detection+of+products.">Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.</a> Nature Protocols. 3 (5), 877-882 (2008).</li><li>Li, Y., Fan, P., Zhou, S., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+A+novel+rapid+detection+platform+for+pathogens.">Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens.</a> Microbial Pathogenesis. 107, 54-61 (2017).</li><li>Notomi, T., Mori, Y., Tomita, N., Kanda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+principle,+features,+and+future+prospects.">Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects.</a> Journal of Microbiology. 53 (1), 1-5 (2015).</li><li>Augustine, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+A+rapid,+sensitive,+specific,+and+cost-effective+point-of-care+test+for+coronaviruses+in+the+context+of+COVID-19+pandemic.">Loop-mediated isothermal amplification (LAMP): A rapid, sensitive, specific, and cost-effective point-of-care test for coronaviruses in the context of COVID-19 pandemic.</a> Biology (Basel). 9 (8), 182 (2020).</li><li>. Nextstrain Available from: <a target="_blank" href="https://nextstrain.org/">https://nextstrain.org/</a> (2023)</li><li>. Neb Lamp, NEB LAMP Available from: <a target="_blank" href="https://lamp.neb.com/">https://lamp.neb.com/</a> (2023)</li><li>. Blast: Basic local alignment search tool (no date) Available from: <a target="_blank" href="https://blast.ncbi.nlm.nih.gov/">https://blast.ncbi.nlm.nih.gov/</a> (2023)</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+molecular+detection+of+SARS-CoV-2+(COVID-19)+virus+RNA+using+colorimetric+LAMP.">Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP.</a> medRxiv. , (2020).</li><li>Lu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+reverse+transcription+loop-mediated+isothermal+amplification+method+for+rapid+detection+of+SARS-CoV-2.">Development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2.</a> Virologica Sinica. 35 (3), 344-347 (2020).</li><li>Najafov, A., Hoxhaj, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PCR Guru. , (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+colorimetric+loop-mediated+isothermal+amplification+speed+and+sensitivity+with+guanidine+chloride.">Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride.</a> Biotechniques. 69 (3), 178-185 (2020).</li><li>Ram&#237;rez-Chavarr&#237;a, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+analysis+of+isothermal+amplification+via+impedance+time-constant-domain+spectroscopy:+A+SARS-CoV-2+case+study.">Automatic analysis of isothermal amplification via impedance time-constant-domain spectroscopy: A SARS-CoV-2 case study.</a> Chemosensors. 11 (4), 230 (2023).</li><li>Haque, M. F. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+RdRp-based+colorimetric+RT-LAMP+assay+for+rapid+and+sensitive+detection+of+SARS-CoV-2+in+clinical+and+sewage+samples+from+Pakistan.">A novel RdRp-based colorimetric RT-LAMP assay for rapid and sensitive detection of SARS-CoV-2 in clinical and sewage samples from Pakistan.</a> Virus Research. 302, 198484 (2021).</li><li>Donia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+RT-LAMP+and+microfluidic+technology+for+detection+of+SARS-CoV-2+in+wastewater+as+an+advanced+point-of-care+platform.">Integration of RT-LAMP and microfluidic technology for detection of SARS-CoV-2 in wastewater as an advanced point-of-care platform.</a> Food and Environmental Virology. 14, 364-373 (2022).</li></ol>

Selv om RT-LAMP blir sett på som en komplementær metodikk for å utføre molekylær diagnostikk, har den også noen begrensninger og kritiske trinn som må vurderes når protokollen standardiseres og implementeres.
LAMP-standardiseringen for påvisning av SARS-CoV-2 evaluerte følgende parametere og komponenter i masterblandingen: (a) konsentrasjon og temperatur for justering av primerne; (b) konsentrasjon av enzymene; (c) magnesiumkonsentrasjon; (d) reaksjonstid; (e) inkludering av tilsetni...

Det alvorlige akutte respiratoriske syndromet coronavirus 2 (SARS-CoV-2) forårsaker koronavirussykdom 2019 (COVID-19). Verdens helseorganisasjon erklærte en folkehelsekrise av internasjonal bekymring 30 januar 2020 og en pandemi 11 mars 2020. Pandemien resulterte i over 760 millioner tilfeller og 6,87 millioner dødsfall per datoen denne artikkelen ble skrevet1.
Virkningen av dette viruset har fremhevet behovet for bedre, mer nøyaktige, raskere og mer allment tilgjengelige overvåkingsverktøy for å forbedre påvisning og kontroll av smittsomme sykdommer 2,3. Under pandemien var SARS-CoV-2 diagnostiske tester basert på påvisning av nukleinsyre, antistoffer og proteiner, men RT-PCR-deteksjon av nukleinsyre er gullstandarden4. RT-PCR har imidlertid noen begrensninger; Det krever spesialisert utstyr, infrastruktur og personell som er opplært i molekylærbiologi, og begrenser bruken til spesialiserte laboratorier. Videre er det tidkrevende (4-6 timer), ikke inkludert tiden for å transportere prøvene til laboratoriet, noe som kan ta dager5. Disse begrensningene hindrer effektiv prøvebehandling og innhenting av informasjonen som kreves for beredskapsplanlegging og epidemiologisk behandling.
Omvendt transkripsjonssløyfemediert isotermisk amplifikasjon (RT-LAMP) har flere fordeler fremfor RT-PCR, noe som gjør det til en tiltalende strategi for å designe fremtidige pasientnære diagnostiske tester (POCT), spesielt i ressursbegrensede omgivelser6. For det første er den veldig spesifikk fordi den bruker mellom fire og seks primere som gjenkjenner seks til åtte områder i målsekvensen, det være seg DNA eller RNA 7,8. For det andre, fordi den opererer ved en konstant temperatur, krever den ikke sofistikert utstyr som sanntids termiske syklister for å generere forsterkningen, og det krever heller ikke høyt utdannet personell for å betjene den. For det tredje er reaksjonstiden svært kort (~60 min), og reagenser som ikke er veldig spesialiserte brukes, noe som gjør det til et kostnadseffektivt verktøy6. Gitt det foregående og helsekrisen forårsaket av COVID-19-pandemien, kan denne teknikken sees på som en alternativ diagnostisk metode som er rask, billig og enkel å implementere i ethvert forskningslaboratorium9.
Protokollen for standardisering og implementering av en RT-LAMP for å oppdage SARS-CoV-2 ved kolorimetriske metoder ved bruk av en termosyklist og et vannbad er beskrevet i denne artikkelen (figur 1). Kritiske punkter, deres begrensninger og alternativer for å fremme dem diskuteres.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65662/65662fig01.jpg"> Figur 1: Skjema for protokollen for forsterkning av SARS-CoV-2 ved hjelp av RT-LAMP-teknikken. Klikk <a href="https://www.jove.com/files/ftp_upload/65662/65662fig01large.jpg" target="_blank">her for å se en større versjon av denne figuren.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 kb DNA Ladder</td>
			<td>SOLIS BIODYNE</td>
			<td>07-12-00050</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>50x TAE Electrophoresis Buffer</td>
			<td>ThermoScientific</td>
			<td>B49</td>
			<td>Store at roome temperature</td>
		</tr>
		<tr>
			<td>Accuris High Fidelity Polymerase</td>
			<td>ACCURIS LIFE SCIENCE REAGENTS</td>
			<td>PR1000-HF-200</td>
			<td>It can be used in case Q5 High-Fidelity DNA polymerase cannot be purchased. For the enzyme, make aliquots of an appropriate volume and store at -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>PanReacAppliChem</td>
			<td>A8963,0100</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Bst 3.0 DNA Polymerase 8000 IU/mL</td>
			<td>New England BioLabs</td>
			<td>M0374S/M0374L</td>
			<td>For the enzyme, make aliquots of an appropriate volume and store at -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Set</td>
			<td>New England BioLabs</td>
			<td>N0446S</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>159220-25G&#160;</td>
			<td>Handle it with caution under an extraction cabinet</td>
		</tr>
		<tr>
			<td>GeneRuler 100 bp Plus DNA Ladder, ready-to-use</td>
			<td>ThermoScientific</td>
			<td>SM0322</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hydroxy naphthol blue disodium salt</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-215156B</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Q5 High-Fidelity DNA polymerase 2000 IU/mL&#160;</td>
			<td>New England BioLabs</td>
			<td>M0491S/M0491L</td>
			<td>For the enzyme, make aliquots of an appropriate volume and store at -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>WarmStart RTx Reverse Transcriptase 15000 IU/mL</td>
			<td>New England BioLabs</td>
			<td>M0380S/M0380L</td>
			<td>For the enzyme, make aliquots of an appropriate volume and store at -20 &#176;C until use.</td>
		</tr>
	</tbody>
</table>

detecting sars-cov-2 virus by reverse transcription-loop-mediated isothermal amplification

Det alvorlige akutte respiratoriske syndromet coronavirus 2 (SARS-CoV-2)-viruset har dramatisk påvirket menneskers helse. Det fortsetter å være en trussel mot det moderne samfunnet fordi mange mennesker dør som følge av infeksjon. Sykdommen diagnostiseres ved hjelp av serologiske og molekylære tester, for eksempel gullstandarden sanntids polymerasekjedereaksjon (RT-PCR). Den siste har flere ulemper fordi den krever spesialisert infrastruktur, kostbart utstyr og opplært personell. Her presenterer vi en protokoll som skisserer trinnene som kreves for å oppdage SARS-CoV-2-viruset ved bruk av omvendt transkripsjonssløyfemediert isotermisk amplifikasjon (RT-LAMP) i humane prøver. Protokollen inkluderer instruksjoner for design av primere i silico, klargjøring av reagenser, amplifikasjon og visualisering. Når den er standardisert, kan denne metoden enkelt implementeres og tilpasses ethvert laboratorium eller behandlingssted innen 60 minutter til en lav pris og ved hjelp av billig utstyr. Den kan tilpasses til å oppdage forskjellige patogener. Dermed kan den potensielt brukes i felten og i helsesentre for å utføre rettidig epidemiologisk overvåking.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The World Health Organization declared a public health emergency of international concern on 30 January 2020 and a pandemic on 11 March 2020. The pandemic resulted in over 760 million cases and 6.87 million deaths as of the date this article was written1.
The impact of this virus has highlighted the need for better, more accurate, faster, and more widely available surveillance tools to improve infectious disease detection and control2,3. During the pandemic, SARS-CoV-2 diagnostic tests were based on detecting nucleic acid, antibodies, and proteins, but RT-PCR detection of nucleic acid is the gold standard4. However, RT-PCR has some limitations; it requires specialized equipment, infrastructure, and personnel trained in molecular biology, limiting its application to specialized laboratories. Further, it is time-consuming (4-6 h), not including the time to transport the specimens to the laboratory, which can take days5. These constraints prevent efficient sample processing and obtaining the information required for contingency planning and epidemiological management.
Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) has several advantages over RT-PCR, making it an appealing strategy for designing future point-of-care diagnostic tests (POCT), particularly in resource-constrained settings6. First, it is greatly specific because it uses between four and six primers that recognize six to eight areas in the target sequence, be it DNA or RNA7,8. Second, because it operates at a constant temperature, it does not require sophisticated equipment such as real-time thermal cyclers to generate the amplification, nor does it necessitate highly trained personnel to operate it. Third, the reaction time is very short (~60 min), and reagents that are not very specialized are employed, which makes it a cost-effective tool6. Given the foregoing and the health emergency caused by the COVID-19 pandemic, this technique can be viewed as an alternative diagnostic method that is quick, inexpensive, and simple to implement in any research laboratory9.
The protocol for standardizing and implementing an RT-LAMP to detect SARS-CoV-2 by colorimetric methods using a thermocycler and a water bath is described in this article (Figure 1). Critical points, their limitations, and alternatives to advance them are discussed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65662/65662fig01.jpg" /> 
Figure 1: Scheme of the protocol for amplifying SARS-CoV-2 using the RT-LAMP technique. <a href="https://www.jove.com/files/ftp_upload/65662/65662fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Forfatter Spotlight: Fremme patogendiagnostikk med standardisert LAMP

Oppdage SARS-CoV-2-virus ved omvendt transkripsjonssløyfemediert isotermisk amplifikasjon

Our research aims to standardize LAMP as a prediagnostic test, suitable for field or primary care settings lacking advanced laboratory infrastructure. We seek to promote the use of locally-producible buffers and alternative dyes, reducing dependence on imported commercial kits. Currently, CRISPR-Cas is an efficient system employed in the development of fast, specific, and sensitive diagnostic methods for pathogens.Now, this system has a broad range of and supplements to detect RNA or DNA targets in different samples of interest. The current experimental challenges involve preventing non-target amplification resulting from amplicon contamination and primary cell especially with lengthy primers prone to dimer formation. Additionally, we aim to determine optimal reaction conditions to achieve a visually distinguishable color change in the dye.Once it's standardized and fully validated, this method can be easily implemented and adapt to any laboratory or point-of-care within 60 minutes, at low cost and using inexpensive equipment, to detect almost any pathogen. Thus, it can potentially be used in primary care centers to carry out timely, epidemiological surveillance. Our multidisciplinary group is focused on the discovery of molecules from native Colombia plants, trying to identify the potential to inhibit the replication of viruses such as dengue, Zika, or SARS-COVID-2.In addition, we are conducting studies to develop molecular diagnostic methods for these viruses.

Implementeringen av protokollen starter med å designe settet med primere for hvert målgen etter protokollen beskrevet ovenfor. I juni 2020 ble 5,000 SARS-CoV-2-genomer hentet fra NextStrain databasen, med en 10 % representativitet av colombianske genomer. Disse sekvensene ble justert for å oppnå konsensussekvensen som ble brukt i primerdesignprosessen. Tabell 1 viser primersettet som er valgt for primerne RdRp/Hel og RdRp. Primersettet for gen N-amplifikasjon ble hentet fra en tidligere publisert rap...

Read this Scientific Article Protocol about Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP at JoVE.com

Her gir vi en komplett protokoll for å standardisere og implementere metoden for å oppdage SARS-CoV-2-viruset i humane prøver ved omvendt transkripsjonssløyfemediert isotermisk amplifikasjon (RT-LAMP). Denne metoden, utført innen 60 minutter, kan tilpasses ethvert laboratorium eller behandlingssted til en lav pris og ved hjelp av billig utstyr.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern ...

Vår forskning tar sikte på å standardisere LAMP som en prediagnostisk test, egnet for felt- eller primærhelsetjenester som mangler avansert laboratorieinfrastruktur. Vi søker å fremme bruken av lokalt produserbare buffere og alternative fargestoffer, og redusere avhengigheten av importerte kommersielle sett. For tiden er CRISPR-Cas et effektivt system som brukes i utviklingen av raske, spesifikke og sensitive diagnostiske metoder for patogener.Nå har dette systemet et bredt spekter av og kosttilskudd for å oppdage RNA- eller DNA-mål i forskjellige prøver av interesse. De nåværende eksperimentelle utfordringene involverer å forhindre ikke-målamplifikasjon som følge av amplikontaminering og primærcelle, spesielt med lange primere som er utsatt for dimerdannelse. I tillegg tar vi sikte på å bestemme optimale reaksjonsforhold for å oppnå en visuelt gjenkjennelig fargeendring i fargestoffet.Når den er standardisert og fullstendig validert, kan denne metoden enkelt implementeres og tilpasses ethvert laboratorium eller behandlingssted innen 60 minutter, til lave kostnader og ved hjelp av billig utstyr, for å oppdage nesten alle patogener. Dermed kan den potensielt brukes i primærhelsesentre for å utføre rettidig, epidemiologisk overvåking. Vår tverrfaglige gruppe er fokusert på oppdagelsen av molekyler fra innfødte Colombia-planter, og prøver å identifisere potensialet for å hemme replikasjonen av virus som dengue, Zika eller SARS-COVID-2.I tillegg gjennomfører vi studier for å utvikle molekylære diagnostiske metoder for disse virusene.

detecting-sars-cov-2-virus-reverse-transcription-loop-mediated

Research

JoVE Journal

Biology

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.

Here we provide a complete protocol to standardize and implement the method for detecting the SARS-CoV-2 virus in human samples by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method, done within 60 min, could be adapted to any laboratory or point-of-care at a low cost and using inexpensive equipment.

Reverse Transcriptase-Loop Mediated Isothermal Amplification Reaction (RT-LAMP) for SARS-CoV-2 Detection

To begin, switch on the laminar airflow cabinet, allowing it to stabilize for three minutes. Aseptically clean and sanitize the cabinet's internal surfaces using the following disinfection agents. After disinfecting any material that will enter the cabinet, place napkins and nitrile gloves into the cabinet.Then switch off the cabinet and expose it to ultraviolet light for 15 minutes. After the irradiation, restart the cabinet. Place the primer design reagents in an ice-filled cooler.After sanitizing it with 70%ethanol, place the container inside the cabinet. Prepare the loop mediated isothermal amplification, or LAMP mix, in a 0.6 milliliter microcentrifuge tube. Pipette the mixture to homogenize it.Next, incubate the closed tubes in a heating block at 95 degrees Celsius for five minutes. Then cool the tubes in an ice-filled polystyrene cooler for five minutes. After cooling, place the tubes in the laminar flow cabinet.To complete the LAMP mix, add four microliters of DNA polymerase, followed by one microliter of reverse transcriptase. To perform colorimetric detection, add the dihydroxynaphthol blue. After reagent addition, pipette each solution to properly mix the LAMP reagents.Next, pipette 22 microliters of the solution into PCR tubes, being careful not to create bubbles. For the negative control, add three microliters of 0.1%diethyl pyrocarbonate water. Keep aside any remaining tubes for the addition of genetic material.Next, remove all materials from the cabinet. After sanitizing the cabinet surfaces with 70%ethanol, shut it off. In a separate area, add three microliters of the sample to each PCR tube and pipette well with a 20 microliter micropipette to thoroughly homogenize.Before initiating the colorimetric reaction, use a high quality camera to take pictures of the PCR tubes. Now, carry out the reaction using a thermal cycler and a water bath. For the thermal cycler, place the tubes into the reaction block and set up thermo profile on the equipment.If using the water bath, securely place the tubes in circular containers, then place these containers in the water bath at 66.3 degrees Celsius for 60 minutes. After the reaction time, remove the tubes and store them at four degrees Celsius until use. If a colorimetric reaction was performed, capture images of the PCR with a high quality camera.A temperature gradient demonstrated that the optimal melting temperature was 66.3 degrees Celsius. The concentration of BST 3.0 enzyme was optimized to 3.2 international units per microliter. To define the colorimetric strategy, phenol red in neutral red dyes were evaluated.However, no color change was observed after amplification. Standardization of different concentrations of hydroxynaphthol blue was performed with the optimum change occurring with 125 micromoles of hydroxynaphthol blue. The amplified samples demonstrated a change in the color in accordance with their concentration.Sample amplification was further confirmed by electrophoresis.