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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 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 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 pan....
The samples used were provided by the clinical laboratory of Fundación 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 Fundación Valle del Lili University hospital.
1. RT-LAMP primer design and preparation
NOTE: L.......
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.......
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.......
This work was funded by Sistema General de Regalías 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 Fundación Valle del Lili for viral RNA from Sars -CoV-2 samples and Dr. Alvaro Barrera-Ocampo for the comments on the manuscript.
....Name | Company | Catalog Number | Comments |
1 kb DNA Ladder | SOLIS BIODYNE | 07-12-00050 | Store at -20 °C |
50x TAE Electrophoresis Buffer | ThermoScientific | B49 | Store at roome temperature |
Accuris High Fidelity Polymerase | ACCURIS LIFE SCIENCE REAGENTS | PR1000-HF-200 | 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 °C until use. |
Agarose | PanReacAppliChem | A8963,0100 | N/A |
Bst 3.0 DNA Polymerase 8000 IU/mL | New England BioLabs | M0374S/M0374L | For the enzyme, make aliquots of an appropriate volume and store at -20 °C until use. |
Deoxynucleotide (dNTP) Solution Set | New England BioLabs | N0446S | Store at -20 °C |
Diethyl pyrocarbonate | Sigma-Aldrich | 159220-25G | Handle it with caution under an extraction cabinet |
GeneRuler 100 bp Plus DNA Ladder, ready-to-use | ThermoScientific | SM0322 | Store at -20 °C |
Hydroxy naphthol blue disodium salt | Santa Cruz Biotechnology | sc-215156B | N/A |
Q5 High-Fidelity DNA polymerase 2000 IU/mL | New England BioLabs | M0491S/M0491L | For the enzyme, make aliquots of an appropriate volume and store at -20 °C until use. |
WarmStart RTx Reverse Transcriptase 15000 IU/mL | New England BioLabs | M0380S/M0380L | For the enzyme, make aliquots of an appropriate volume and store at -20 °C until use. |
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