Name: Author Spotlight: Advancing Pathogen Diagnostics with Standardized 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

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: L...

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>

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

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.

Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP

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

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.

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

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.

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

Research

JoVE Journal

Biology

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.