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

Summary

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.

Abstract

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.

Introduction

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 control^2,3. 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 RNA^7,8. 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.

Figure 1: Scheme of the protocol for amplifying SARS-CoV-2 using the RT-LAMP technique. Please click here to view a larger version of this figure.

Protocol

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…

Representative Results

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…

Discussion

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…

Materials

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.