Name: Forfatter Spotlight: Fremme af patogendiagnostik med standardiseret 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 arbejde blev finansieret af Sistema General de Regalías fra Colombia, bevillingsnummer BPIN 2020000100092, og Universidad Icesi - Convocatoria Interna, bevillingsnummer CA0413119. MFVT blev også finansieret af adjunktmidlerne fra Universidad de los Andes. Bevillingsgiverne deltog ikke i design, udførelse af aktiviteter, dataindsamling og dataanalyse og udarbejdelse af manuskriptet. Vi takker University Hospital Fundación Valle del Lili for viralt RNA fra Sars-CoV-2-prøver og Dr. Alvaro Barrera-Ocampo for kommentarerne til manuskriptet.

De anvendte prøver blev leveret af det kliniske laboratorium på Fundación Valle del Lili Universitetshospital og svarede til det oprensede RNA fra patienter, der testede positive for COVID-19 ved hjælp af RT-qPCR-teknikken. Alle patienter gav informeret samtykke til forskning, og denne undersøgelse blev godkendt af den bioetiske komité for menneskelige undersøgelser fra Fundación Valle del Lili Universitetshospital.
1. RT-LAMP primer design og forberedelse
<p class="...

Brug af kolorimetrisk og RT-LAMP-analyse til at detektere 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>

Selvom RT-LAMP betragtes som en komplementær metode til udførelse af molekylær diagnostik, har den også nogle begrænsninger og kritiske trin, der skal overvejes, når protokollen standardiseres og implementeres.
LAMP-standardiseringen til detektion af SARS-CoV-2 evaluerede følgende parametre og komponenter i masterblandingen: (a) koncentration og temperatur for justering af primerne; b) koncentrationen af enzymerne c) magnesiumkoncentration d) reaktionstid e) tilsætningsstoffer som BSA,...

Det alvorlige akutte respiratoriske syndrom coronavirus 2 (SARS-CoV-2) forårsager coronavirussygdom 2019 (COVID-19). Verdenssundhedsorganisationen erklærede en folkesundhedsmæssig krisesituation af international betydning den 30. januar 2020 og en pandemi den 11. marts 2020. Pandemien resulterede i over 760 millioner tilfælde og 6,87 millioner dødsfald på den dato, hvor denne artikel blev skrevet1.
Virkningen af denne virus har understreget behovet for bedre, mere præcise, hurtigere og mere bredt tilgængelige overvågningsværktøjer for at forbedre påvisning og kontrol af infektionssygdomme 2,3. Under pandemien var SARS-CoV-2 diagnostiske tests baseret på påvisning af nukleinsyre, antistoffer og proteiner, men RT-PCR-påvisning af nukleinsyre er guldstandarden4. RT-PCR har dog nogle begrænsninger; Det kræver specialiseret udstyr, infrastruktur og personale uddannet i molekylærbiologi, hvilket begrænser dets anvendelse til specialiserede laboratorier. Yderligere er det tidskrævende (4-6 timer), ikke medregnet tiden til at transportere prøverne til laboratoriet, hvilket kan tage dage5. Disse begrænsninger forhindrer effektiv prøvebehandling og opnåelse af de oplysninger, der er nødvendige for beredskabsplanlægning og epidemiologisk styring.
Omvendt transkriptionsloop-medieret isotermisk amplifikation (RT-LAMP) har flere fordele i forhold til RT-PCR, hvilket gør det til en tiltalende strategi til design af fremtidige diagnostiske tests (POCT), især i ressourcebegrænsede omgivelser6. For det første er den meget specifik, fordi den bruger mellem fire og seks primere, der genkender seks til otte områder i målsekvensen, det være sig DNA eller RNA 7,8. For det andet, fordi det fungerer ved en konstant temperatur, kræver det ikke sofistikeret udstyr såsom termiske cykler i realtid for at generere forstærkningen, og det kræver heller ikke højtuddannet personale at betjene det. For det tredje er reaktionstiden meget kort (~60 min), og der anvendes reagenser, der ikke er særlig specialiserede, hvilket gør det til et omkostningseffektivt værktøj6. I betragtning af ovenstående og den sundhedsmæssige nødsituation forårsaget af COVID-19-pandemien kan denne teknik ses som en alternativ diagnostisk metode, der er hurtig, billig og enkel at implementere i ethvert forskningslaboratorium9.
Protokollen til standardisering og implementering af en RT-LAMP til detektering af SARS-CoV-2 ved hjælp af kolorimetriske metoder ved hjælp af en termocykler og et vandbad er beskrevet i denne artikel (figur 1). Kritiske punkter, deres begrænsninger og alternativer til at fremme dem diskuteres.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65662/65662fig01.jpg"> Figur 1: Skema over protokollen til amplifikation af SARS-CoV-2 ved hjælp af RT-LAMP-teknikken. Klik <a href="https://www.jove.com/files/ftp_upload/65662/65662fig01large.jpg" target="_blank">her for at se en større version af denne figur.</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

Coronavirus 2 (SARS-CoV-2) virus med alvorligt akut respiratorisk syndrom har haft en dramatisk indvirkning på menneskers sundhed. Det er fortsat en trussel mod det moderne samfund, fordi mange mennesker dør som følge af infektion. Sygdommen diagnosticeres ved hjælp af serologiske og molekylære tests, såsom guldstandarden realtidspolymerasekædereaktion (RT-PCR). Sidstnævnte har flere ulemper, fordi det kræver specialiseret infrastruktur, dyrt udstyr og uddannet personale. Her præsenterer vi en protokol, der skitserer de trin, der kræves for at detektere SARS-CoV-2-virussen ved hjælp af omvendt transkriptionsloop-medieret isotermisk amplifikation (RT-LAMP) i humane prøver. Protokollen indeholder instruktioner til design af primere i silico, fremstilling af reagenser, amplifikation og visualisering. Når den er standardiseret, kan denne metode nemt implementeres og tilpasses ethvert laboratorium eller behandlingssted inden for 60 minutter til en lav pris og ved hjælp af billigt udstyr. Det kan tilpasses til at påvise forskellige patogener. Således kan det potentielt bruges i marken og i sundhedscentre til at udføre rettidig epidemiologisk overvågning.

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 af patogendiagnostik med standardiseret LAMP

Påvisning af SARS-CoV-2-virus ved omvendt transkriptions-loop-medieret isotermisk amplifikation

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 af protokollen starter med at designe sættet af primere for hvert målgen efter protokollen beskrevet ovenfor. I juni 2020 blev 5.000 SARS-CoV-2-genomer opnået fra NextStrain databasen med en 10 % repræsentativitet af colombianske genomer. Disse sekvenser blev justeret for at opnå den konsensussekvens, der blev brugt i primerdesignprocessen. Tabel 1 viser det primersæt, der er valgt for primerne RdRp/Hel og RdRp. Primersættet for gen N-amplifikation blev opnået fra en tidligere of...

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

Her giver vi en komplet protokol til at standardisere og implementere metoden til påvisning af SARS-CoV-2-virussen i humane prøver ved omvendt transkriptionsloop-medieret isotermisk amplifikation (RT-LAMP). Denne metode, der udføres inden for 60 minutter, kan tilpasses ethvert laboratorium eller behandlingssted til en lav pris og ved hjælp af billigt udstyr.

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

Vores forskning har til formål at standardisere LAMP som en prædiagnostisk test, der er velegnet til felt- eller primærplejemiljøer, der mangler avanceret laboratorieinfrastruktur. Vi søger at fremme brugen af lokalt producerbare buffere og alternative farvestoffer, hvilket reducerer afhængigheden af importerede kommercielle kits. I øjeblikket er CRISPR-Cas et effektivt system, der anvendes til udvikling af hurtige, specifikke og følsomme diagnostiske metoder for patogener.Nu har dette system en bred vifte af og kosttilskud til at detektere RNA- eller DNA-mål i forskellige prøver af interesse. De nuværende eksperimentelle udfordringer involverer forebyggelse af ikke-målamplifikation som følge af amplikontaminering og primær celle, især med lange primere, der er tilbøjelige til dimerdannelse. Derudover sigter vi mod at bestemme optimale reaktionsbetingelser for at opnå en visuelt skelnelig farveændring i farvestoffet.Når den er standardiseret og fuldt valideret, kan denne metode nemt implementeres og tilpasses ethvert laboratorium eller behandlingssted inden for 60 minutter, til lave omkostninger og ved hjælp af billigt udstyr til at opdage næsten ethvert patogen. Således kan det potentielt bruges i primære plejecentre til at udføre rettidig, epidemiologisk overvågning. Vores tværfaglige gruppe er fokuseret på opdagelsen af molekyler fra indfødte colombianske planter og forsøger at identificere potentialet til at hæmme replikationen af vira som dengue, Zika eller SARS-COVID-2.Derudover udfører vi undersøgelser for at udvikle molekylære diagnostiske metoder for disse vira.

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.