This work was funded by Sistema General de Regal&#237;as of Colombia, grant number BPIN 2020000100092, and Universidad Icesi - Convocatoria Interna, grant number CA0413119. MFVT was also financed by the Assistant Professorship Funds from Universidad de los Andes. The funding entities did not participate in the design, execution of activities, data collection, and data analysis and preparation of the manuscript. We thank to University Hospital Fundaci&#243;n Valle del Lili for viral RNA from Sars -CoV-2 samples and Dr. Alvaro Barrera-Ocampo for the comments on the manuscript.

The samples used were provided by the clinical laboratory of Fundaci&#243;n Valle del Lili University hospital and corresponded to the purified RNA from patients who tested positive for COVID-19 using the RT-qPCR technique. All patients provided informed consent for research, and this study was approved by the bioethical committee for human studies from Fundaci&#243;n Valle del Lili University hospital.
1. RT-LAMP primer design and preparation
NOTE: LAMP primers can be used with a variety of platforms, including New England BioLabs (NEB) LAMP, Primer Explorer, and LAMP assay versatile analysis (LAVA). However, for this protocol, the NEB LAMP tool was used. Primer design can be done using SARS-CoV-2 genomes obtained from NextStrain database10. Table 1 shows the primer set used in this protocol.
<ol>
	<li>Primer design for LAMP
	<ol>
		<li>Obtain viral genome sequences.</li>
		<li>Perform sequence alignments to obtain the consensus sequence.</li>
		<li>Navigate to the NEB LAMP primer design tool platform11&#160;and follow the instructions in the quick guide. This tool produces the same results as primer explorer V5, but it is much more user-friendly in its output. Use primer explorer user manuals as a guide for primer design.</li>
	</ol>
	</li>
	<li>Thermodynamic evaluation of the set of primers
	<ol>
		<li>Use the tool Primer-Dimer12 to perform a thermodynamic analysis on the primers obtained.</li>
		<li>Put the primer sequences in the tool. Then, select the option Multiplex Analysis and Dimer Structure Report.</li>
		<li>Select the primer sets that have &#916;G not less than -5.</li>
	</ol>
	</li>
	<li>Specificity evaluation of the designed primers
	<ol>
		<li>Use the Nucleotide collection (nt/nt) database in BLAST13 to analyze each primer.</li>
		<li>To perform the first BLAST analysis, select the Refseq_rna Data Base and filter the search with the group of genera that belong to the subfamily Orthocoronavirinae. They are Alphacoronavirus (taxid:693996), Gammacoronavirus (taxid:694013), and Deltacoronavirus (taxid:1159901). Additionally, evaluate the sequence against other viruses that are co-circulating as H1N1 subtype (taxid:114727), Influenza A virus (taxid:11320), and Influenza B virus (taxid:11520).</li>
		<li>To perform the second BLAST analysis, select the Betacoronavirus GenBank and filter the search with Coronaviridae (taxid:11118) and SARS (taxid:694009). These groups contain sequences of all identified SARS Coronavirus genomes, including genomes found in bats, Betacoronavirus (taxid:694002).</li>
		<li>For this protocol, ensure that the primers do not align with genomes other than the target genome, SARS-CoV-2.</li>
	</ol>
	</li>
	<li>Primer preparation
	<ol>
		<li>Spin the vials containing the Iyophilized primers with a microcentrifuge (10,000 x g, 1 min at room temperature [RT]) to avoid losses during tube opening.</li>
		<li>Rehydrate the Iyophilized&#160; powder in 0.1% diethyl pyrocarbonate (DEPC) water or nuclease-free water to a final concentration of 100 &#181;M (Table 2) and thoroughly dissolve by pipetting up and down. Then, spin at maximum speed (10,000 x g, 1 min at RT) in a microcentrifuge to collect all of the primer solutions at the bottom of the tube.</li>
		<li>Prepare the 10x primer mix under a biosafety cabinet with the forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3), backward outer primer (B3), loop backward (LB), and loop forward (LF) primers, as reported in Table 2. To prevent losses, pipette or gently vortex the primer solution before performing a rapid spin (10,000 x g, 1 min at RT) with a microcentrifuge.</li>
		<li>Store the 10x primer mix at &#8722;20 &#176;C for long-term storage; however, prepare enough for a maximum of five experiments, regardless of several samples to avoid too many freeze-thaw cycles. 
		NOTE If a smaller volume of the primer mix is needed, then adjust the values by calculating the new volumes (Table 2). Furthermore, the RdRp and RdRp/Hel sets do not include the LF primer because loop primers are not required for RT-LAMP reactions. As a result, replace the volume of LF primer with nuclease-free water or 0.1% DEPC water.</li>
	</ol>
	</li>
</ol>
2. RT-LAMP reaction
<ol>
	<li>Turn on the laminar flow cabinet according to the manufacturer&#39;s instructions and wait for at least 3 min for the airflow to stabilize.</li>
	<li>Once the airflow is stable, clean and sanitize the internal surfaces of the cabinet using an aseptic technique. To accomplish this, use the following disinfectants in this order: 1000 ppm quaternary ammonium (benzalkonium chloride), 2% hypochlorite, 3% hydrogen peroxide, and 70% ethanol. 
	NOTE: In this case, the aseptic technique entails applying the disinfectant and removing it with napkins from inside the cabin to the outside without going over previously cleaned surfaces.</li>
	<li>Using the disinfectants from step 2.2, clean the materials that will enter the cabin in the same order. 
	NOTE: Micropipettes, filter tip boxes, flasks with 1.5 mL and 0.6 mL tubes, 0.2 mL PCR tubes, racks, and a 400 mL beaker must be brought into the cabinet.</li>
	<li>Bring some napkins and nitrile gloves into the cabin. After that, turn off the cabinet and expose it to ultraviolet (UV) light for 15 min. 
	CAUTION: To avoid tissue and DNA damage from prolonged radiation exposure, avoid UV light until the time set in step 2.4 expires. 
	NOTE Perform the assembly shown in Figure 2 before beginning the protocol, and begin the water bath after completing step 2.4. It is crucial to fill the metal container almost to the brim with drinking water and set the temperature of the iron laboratory heating plate to 90 &#176;C, as this will result in a temperature of ~66.3 &#176;C in the system, which is monitored with the mercury thermometer.</li>
	<li>After the irradiation period has ended, restart the cabinet and follow the recommendations in step 1.1.</li>
	<li>Place the reagents (Table 3, Table 4, and Table 5) in an ice-filled cooler or small polystyrene refrigerator. Put the container within the cabinet after cleaning it with 70% ethanol.</li>
	<li>In a 0.6 mL microcentrifuge tube, prepare the LAMP mix of the gene to be amplified (RdRp, N-A, and RdRp/Hel), adding only the following components: 10x Buffer, MgSO4, dNTPs, 1x primers mix and nuclease-free water or 0.1% DEPC water; mix well by pipetting to homogenize. 
	CAUTION: Because of improper handling and behavior inside the cabinet, there is a high risk of reagent contamination. The following rules must be followed to mitigate this problem: (i) use sterile and filter tips; (ii) use one tip for each reagent; (iii) move slowly and carefully to avoid disrupting the laminar flow; (iv) keep order and use the fewest materials; and (v) use different gloves to prepare the mix and add the genetic material. 
	NOTE: Keep all the reagents, especially enzymes, on ice because temperature changes can denature them and alter polymerase activity.</li>
	<li>Place the 0.6 mL tube(s) with the cap closed in a heating block and incubate at 95 &#176;C for 5 min. 
	NOTE: Turn on the heating block for 1.5-2.0 mL tubes located outside the cabinet for at least 30 min before beginning the LAMP mix preparation and monitor the temperature (95 &#176;C) with a mercury or alcohol thermometer.</li>
	<li>When the incubation is complete, place the tubes in an ice-filled polystyrene cooler for 5 min.</li>
	<li>Return the tubes to the laminar flow cabinet and complete the LAMP mix preparation by adding the enzymes DNA polymerase (Bst 3.0), reverse transcriptase, and high-fidelity DNA polymerase (Table 3, Table 4, and Table 5). In the case of using colorimetric detection, add the dye hydroxinaphthol blue (HNB).</li>
	<li>After adding these reagents, mix the LAMP reagents very well by pipetting them to solubilize the enzymes and dye.</li>
	<li>Fill each PCR tube with 22.0 &#181;L of the mix, being careful not to create bubbles. Then, add 3.0 &#181;L of 0.1% DEPC water or nuclease-free water to the negative control or tube no template control (NTC) and set aside the remaining tube(s) for the addition (genetic material). 
	NOTE : Keep the PCR tubes in an ice-filled cooler until the sample is added to avoid activating the Bst 3.0 enzyme and starting the reaction prematurely.</li>
	<li>Remove all materials from the cabinet and use 70% ethanol to clean the surfaces. Then turn it off following the manufacturer&#39;s instructions.</li>
	<li>In a separate area, add 3 &#181;L of the sample to each PCR tube and thoroughly homogenize it. Use a 20 &#181;L micropipette and filter tips to accomplish this. 
	CAUTION: The micropipette used to add the genetic material must be used exclusively for this purpose and cannot be used to prepare the mix. In this way, contamination of the reagents is avoided. Additionally, keep RNA samples on the ice at all times to reduce the possibility of RNA degradation. Use the following personal protective equipment (PPE) for the sample addition: disposable gown, cap, N95 mask, leggings, lab goggles, and nitrile gloves.</li>
	<li>Before performing the colorimetric reaction, take photographs of the PCR tubes with a high-quality camera. The starting color with HNB is violet.</li>
	<li>Carry out the reaction in the following system or equipment: (i) thermal cycler and (ii) water bath.
	<ol>
		<li>Thermal cycler: Deposit the tubes into the reaction block and set up the thermoprofile (see Table 6) on the equipment.</li>
		<li>Water Bath: Deposit the tubes in circular containers and adjust them very well to prevent them from coming out. After that, place the containers in the water bath (Figure 2A, B) at the temperature listed in Table 6.</li>
	</ol>
	</li>
	<li>In the case of the water bath, once the tubes are inside the system, start the timer for 60 min (Table 6).</li>
	<li>Remove the tubes from the thermal cycler or water bath after the reaction time and store them at 4 &#176;C for the electrophoretic run or at &#8722;20 &#176;C until use.</li>
	<li>If a colorimetric reaction was performed, take photographs of the PCR tubes using a high-quality camera. The final color with HNB is sky blue.</li>
</ol>
3. Analysis of amplification products in agarose gel
NOTE: These steps are suggested as additional checks to colorimetric reaction or control for performance during the standardization step. This is because the technique could present a huge contamination risk to the lab doing these tests.
<ol>
	<li>Place the bed inside the electrophoresis chamber so that the edge rubbers touch the walls, creating a sealed space for the addition of agarose (internal chamber) (Figure 3A, B).</li>
	<li>After completing step 3.1, weigh the necessary amount of agarose in a 500 mL beaker to obtain a 2% gel. After that, add the required volume of 0.5x Tris-acetate EDTA (TAE) buffer and microwave for 1-2 min. 
	NOTE: The agarose is completely melted when it is translucent and lump-free when removed from the oven. If this is not confirmed, poorly gelled regions may remain, causing the electrophoretic run and visualization of the amplification products to be altered.</li>
	<li>Take the beaker out of the oven and pour the agarose into the internal chamber created in step 3.1 (Figure 3C). Subsequently, check that there are no bubbles, and if there are, remove them using a micropipette tip.</li>
	<li>Arrange the comb to form the wells and leave the agarose to gel for about 30 min at room temperature (RT).</li>
	<li>After this time, add 5 mL of 0.5x TAE buffer to facilitate the removal of the combs and the bed containing the gel. Then position the gel in such a way that the wells are in the anode (Figure 3D).</li>
	<li>Fill the electrophoresis chamber with 0.5x TAE buffer to the capacity specified by the manufacturer, ensuring that the electrodes are in contact with the buffer.</li>
	<li>Add 3 &#181;L of molecular weight marker to the first well of the gel and add 9 &#181;L of NTC and each sample to the following wells. Make these by combining 7 &#181;L of the amplification product with 3 &#181;L of loading buffer; then load 9 &#181;L of this mixture into the gel&#39;s wells.</li>
	<li>Cover the electrophoresis chamber with the lid and connect the cables to the power supply ports in the color pattern. Set the power source to the following parameters: 100 V and constant amperage for 120 min.</li>
	<li>After the electrophoretic run is completed, place the gel in the container with the staining solution (ethidium bromide) and incubate for 30 min.</li>
	<li>After incubation, remove the gel from the staining solution and place it in a zip-lock bag. This prevents contamination of the equipment that will be used to visualize the amplicons.</li>
	<li>Visualize the gel on a transilluminator or imager like the Amersham Imager 600.</li>
</ol>

Using Colorimetric and RT-LAMP Analysis to Detect 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>

Natalia Campillo-Pedroza is CEO of the company BioDx: Diagn&#243;stico y Soluciones Biotecnol&#243;gicas S.A.S. The rest of the authors declare no conflict of interest.

Although the RT-LAMP is regarded as a complementary methodology for performing molecular diagnostics, it also has some limitations and critical steps that must be considered when the protocol is standardized and implemented.
The LAMP standardization for the detection of SARS-CoV-2 evaluated the following parameters and components in the master mix: (a) concentration and temperature of alignment of the primers; (b) concentration of the enzymes; (c) magnesium concentration; (d) reaction time; (e...

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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65662/65662fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>1 kb DNA Ladder</td><td>SOLIS BIODYNE</td><td>07-12-00050</td><td>Store at -20 &amp;deg;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 &amp;deg;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 &amp;deg;C until use.</td></tr><tr><td>Deoxynucleotide (dNTP) Solution Set</td><td>New England BioLabs</td><td>N0446S</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Diethyl pyrocarbonate</td><td>Sigma-Aldrich</td><td>159220-25G&amp;nbsp;</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 &amp;deg;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&amp;nbsp;</td><td>New England BioLabs</td><td>M0491S/M0491L</td><td>For the enzyme, make aliquots of an appropriate volume and store at -20 &amp;deg;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 &amp;deg;C until use.</td></tr></tbody></table>

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.

The implementation of the protocol starts by designing the set of primers for each target gene following the protocol described above. In June 2020, 5,000 SARS-CoV-2 genomes were obtained from the NextStrain database, with a 10% representativeness of Colombian genomes. These sequences were aligned to obtain the consensus sequence that was used in the primer design process. Table 1 shows the primers set chosen for primers RdRp/Hel and RdRp. The primer set for gene N amplification was obtained from a previ...

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

Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP

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.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

593 Views.  Universidad Icesi. 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.

Video: Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

Immunology and Infection

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Multiplex RT-qPCR Assay for Respiratory Virus Detection

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses 

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...

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

Summary

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Chapters in this video

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

The MultiBac Protein Complex Production Platform at the EMBL

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV