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

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.

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: 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.

1. Primer design for LAMP
   1. Obtain viral genome sequences.
   2. Perform sequence alignments to obtain the consensus sequence.
   3. Navigate to the NEB LAMP primer design tool platform¹¹ 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.
2. Thermodynamic evaluation of the set of primers
   1. Use the tool Primer-Dimer¹² to perform a thermodynamic analysis on the primers obtained.
   2. Put the primer sequences in the tool. Then, select the option Multiplex Analysis and Dimer Structure Report.
   3. Select the primer sets that have ΔG not less than -5.
3. Specificity evaluation of the designed primers
   1. Use the Nucleotide collection (nt/nt) database in BLAST¹³ to analyze each primer.
   2. 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).
   3. 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).
   4. For this protocol, ensure that the primers do not align with genomes other than the target genome, SARS-CoV-2.
4. Primer preparation
   1. 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.
   2. Rehydrate the Iyophilized  powder in 0.1% diethyl pyrocarbonate (DEPC) water or nuclease-free water to a final concentration of 100 µM (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.
   3. 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.
   4. Store the 10x primer mix at −20 °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.

2. RT-LAMP reaction

1. Turn on the laminar flow cabinet according to the manufacturer's instructions and wait for at least 3 min for the airflow to stabilize.
2. 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.
3. 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.
4. 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 °C, as this will result in a temperature of ~66.3 °C in the system, which is monitored with the mercury thermometer.
5. After the irradiation period has ended, restart the cabinet and follow the recommendations in step 1.1.
6. 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.
7. 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.
8. Place the 0.6 mL tube(s) with the cap closed in a heating block and incubate at 95 °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 °C) with a mercury or alcohol thermometer.
9. When the incubation is complete, place the tubes in an ice-filled polystyrene cooler for 5 min.
10. 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).
11. After adding these reagents, mix the LAMP reagents very well by pipetting them to solubilize the enzymes and dye.
12. Fill each PCR tube with 22.0 µL of the mix, being careful not to create bubbles. Then, add 3.0 µL 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.
13. Remove all materials from the cabinet and use 70% ethanol to clean the surfaces. Then turn it off following the manufacturer's instructions.
14. In a separate area, add 3 µL of the sample to each PCR tube and thoroughly homogenize it. Use a 20 µL 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.
15. Before performing the colorimetric reaction, take photographs of the PCR tubes with a high-quality camera. The starting color with HNB is violet.
16. Carry out the reaction in the following system or equipment: (i) thermal cycler and (ii) water bath.
    1. Thermal cycler: Deposit the tubes into the reaction block and set up the thermoprofile (see Table 6) on the equipment.
    2. 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.
17. In the case of the water bath, once the tubes are inside the system, start the timer for 60 min (Table 6).
18. Remove the tubes from the thermal cycler or water bath after the reaction time and store them at 4 °C for the electrophoretic run or at −20 °C until use.
19. 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.

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.

1. 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).
2. 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.
3. 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.
4. Arrange the comb to form the wells and leave the agarose to gel for about 30 min at room temperature (RT).
5. 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).
6. 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.
7. Add 3 µL of molecular weight marker to the first well of the gel and add 9 µL of NTC and each sample to the following wells. Make these by combining 7 µL of the amplification product with 3 µL of loading buffer; then load 9 µL of this mixture into the gel's wells.
8. 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.
9. After the electrophoretic run is completed, place the gel in the container with the staining solution (ethidium bromide) and incubate for 30 min.
10. 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.
11. Visualize the gel on a transilluminator or imager like the Amersham Imager 600.

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 previously published report¹⁴.

The first step in the standardization of the protocol was to avoid NTC amplification. One of the most important parameters that must be verified in this regard is determining the optimal melting temperature (T_m) and Bst 3.0 concentration for the set of primers. A temperature gradient was used to determine the best T_m for the amplification (Figure 4A). For the set of primers used in this protocol, the optimal T_m was 66.3 °C (Figure 4A). Furthermore, different concentrations of the Bst 3.0 enzyme were evaluated, with 3.2 lU/µL being the optimal concentration for that reaction (Figure 4B). The concentrations provided by Lu et al.¹⁵ were used for the remaining reagents (Table 3, Table 4, and Table 5).

Once the T_m and Bst 3.0 optimal concentration was determined, the amplification process was carried out. The patient samples were provided by University Hospital Fundación Valle del Lili. The positive and negative samples were previously amplified using a conventional RT-PCR protocol, and the viral RNA was then used for RT-LAMP amplification using the protocol described here and the conditions listed. The amplification of N, RdRp/Hel, and RdRp genes in the patient samples but not in NTC is shown in Figure 5A,B. Given that the RT-LAMP is an appealing strategy for designing POCT in the future, the amplifications in this protocol were implemented in a conventional thermal cycler and a water bath (Figure 6).

The second step in standardizing the protocol was to define the colorimetric strategy, so phenol red and neutral red were the first dyes evaluated; for its evaluation, different dyes concentration was probed (Figure 7), but no color change was observed after amplification with any of the concentrations tested. This result could be explained by the fact that both dyes are pH indicators, meaning they are sensitive to the pH of the sample, especially if the viral RNA was eluted in Buffer TE, which was the case for the patient samples evaluated with this protocol. Hydroxy Naphthol Blue (HNB) was investigated because its color changes with changes in Mg²⁺ concentration, which would be reduced during the amplification reaction as a cofactor of the polymerase enzyme. In this case, different concentrations of HNB were tested to determine which allowed for the best color change after amplification, which occurs in the reaction with 125 µM of HNB (Figure 8). A complete description of reagents employed in the preparation of the amplification mix for N and RdRp/Hel genes by colorimetric LAMP is included in Table 5.

After determining the best conditions for colorimetric detection, patient samples with varying numbers of viral genomes were amplified. Figure 9 shows the amplification of the samples, in which a change of color occurs in the samples according to the concentration of the viral genome. The amplification results were also visualized using agarose electrophoresis, confirming the amplification of patient samples but not NTC.

Figure 2: Diagram of the water bath used to amplify SARS-CoV-2 genes using the LAMP technique. (A) Front view and (B) top view. Please click here to view a larger version of this figure.

Figure 3: Electrophoresis chamber assembly. (A) Diagram of the electrophoresis chamber used to separate the PCR products. (B) Formation of the internal chamber for the preparation of the agarose gel. (C) Addition of the molten agarose in the internal chamber. (D) Disassembly of the inner chamber to accommodate the gel in the running position. Please click here to view a larger version of this figure.

Figure 4: Standardization of the amplification conditions of the primers and the enzyme Bst 3.0. (A) Each lane shows a different gradient temperature, ranging from 63 °C to 67 °C. From left to right, molecular weight marker (MWM), No Template Control (-), lane 1: 67 °C; lane 2: 66.8 °C; lane 3: 66.3 °C; lane 4: 65.5 °C; lane 5: 64.6 °C; lane 6: 63.9 °C; lane 7: 63.4 °C; lane 8: 63 °C. (B) B1: 8.0 U/µL of Bst 3.0; B2: 6.4 U/µL of Bst 3.0; B3: 4.8 U/µL of Bst 3.0; B4: 3.2 U/µL of Bst 3.0; 1: No Template Control; and 2: patient sample EEDD8 (Ct = 23.39). Please click here to view a larger version of this figure.

Figure 5: Agarose gel electrophoresis of the amplification products of the N and RdRp/Hel genes present in the SARS-CoV-2 virus using the LAMP technique. (A) Replicate 1 and (B) Replicate 2, where MWM: molecular weight marker; 1: No Template Control; 2: patient sample E1123 (Ct = 19.95); 3: patient sample E1324 (Ct = 26.01); 4: patient sample EEDD10 (Ct = 30.09); RH: RdRp/Hel gene and N: N gene. Please click here to view a larger version of this figure.

Figure 6: Agarose gel electrophoresis of the amplification products of the RdRp gene present in the SARS-CoV-2 virus using the LAMP technique. The reaction was carried out in (A) a thermal cycler and (B) a water bath system. MPM: molecular weight marker; 1: No Template Control; 2: patient sample E1123 (Ct = 19.95); 3: patient sample E1757 (Ct = 23.67); 4: patient sample E1604 (Ct = 23.98); 5: patient sample E1245 (Ct = 25.99); 6: patient sample E1324 (Ct = 26.01); 7: patient sample EEDD7 (Ct = 26.56); 8: patient sample 24 (Ct = 37.99) and R: RdRp gene. *Refers to the amplifications that were subjected to 60 min of reaction. Please click here to view a larger version of this figure.

Figure 7: Amplification of the RdRp gene present in the SARS-CoV-2 virus using the LAMP technique with colorimetric detection. Tubes (A) before and (B) after the reaction, where 1: No Template Control; 2: patient sample E1123 (Ct=19.95); 3: patient sample E1757 (Ct=23.67); 4: patient sample E1324 (Ct=26.01); PR: Phenol Red; and NR: Neutral Red. For each dye, four concentrations were evaluated in the final reaction (50 µM, 75 µM, 100 µM, and 120 µM). Please click here to view a larger version of this figure.

Figure 8: Amplification of the RdRp gene present in the SARS-CoV-2 virus by the colorimetric LAMP technique, using the blue hydroxynaphthol indicator. Tubes (A) before and (B) after the reaction, where 1: No Template Control; 2: patient sample E1594 (Ct=20.75); 3: patient sample E990 (Ct=22.67); 4: patient sample E1245 (Ct=25.99). For this dye, four concentrations were evaluated in the final reaction (50 µM, 75 µM, 100 µM, and 125 µM). Please click here to view a larger version of this figure.

Figure 9: Amplification of the samples. Tubes (A) before and (B) after reaction by LAMP using the blue hydroxynaphthol indicator. (C) Agarose gel electrophoresis of the amplification products of the N gene present in the SARS-CoV-2 virus using the colorimetric LAMP technique. MPM: molecular weight marker; NTC: No Template Control; S1: patient sample E1594 (Ct = 20.75); S2: patient sample E990 (Ct = 22.67); S3: patient sample E1245 (Ct = 25.99). Please click here to view a larger version of this figure.

Table 1: Primer sequences for SARS-CoV-2 detection by RT-LAMP.

Table 2: Preparation of 10x RT-LAMP primer mix. The RdRp gene primer mix does not contain the LF primer; therefore, replace this volume with Nuclease-free water. *Instead of Nuclease-free water, 10 mM Tris pH 8.0 prepared in DEPC 0.1% water can be used.

Table 3: Preparation of the RdRp gene amplification mix by LAMP.

Table 4: Preparation of the amplification mix of the N-A and RdRp/Hel genes by LAMP.

Table 5: Preparation of the amplification mix of the N-A and RdRp/Hel genes by colorimetric LAMP.

Table 6: Thermal conditions used for the amplification of the RdRp, N-A, and RdRp/Hel genes by LAMP.

Table 7: Comparison of the dyes used in the visual detection of colorimetric LAMP.

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) inclusion of additives such as BSA, DMSO, Guanidinium Chloride; (f) design of the primers; (g) addition of colorant; (h) use of reaction buffers and recombinant enzymes produced in-house.

This standardization process began with six sets of primers, two reported by Zhang et al.¹⁴ and four designed by the BioDx company. The latter was designed from scratch with available online tools and was selected for its high specificity against SARS-CoV-2, as determined by BLAST. In addition, their thermodynamic evaluation showed a lower tendency to form primer dimers or hairpin structures that favored self-amplification.

Due to the impact that primers have on the standardization of the technique and the results of previous experiments where amplification was found in NTCs, a new primer design was made for the N, S, RdRp, and RdRp/Hel genes. These were subjected to several bioinformatic analyzes to guarantee their specificity and the reduction of the tendency to form self-amplifying structures. The selected primers met the established bioinformatics parameters (ΔG > -5.0 and specificity demonstrated by BLAST).

In silico assays have a very important role in predicting the behavior that can be observed in in vitro reactions since they can be used as an initial filter for the selection of primers. However, these predictions are not the same in all cases; therefore, they cannot be used as a definitive selection parameter but rather as an initial approximation. The actual behavior of the primers in the technique is determined directly during the reaction and visual evaluation by colorimetry or on an agarose gel. Finally, doubtful or unclear results are usually repeated by another analyst or discarded; since an inconclusive result cannot be reported in the context of the diagnosis.

One of the most important critical steps is the high probability of contamination when the protocol is being developed, which is reflected in the amplification of the NTC. This could be avoided by following good laboratory practices such as proper disinfection and cleaning procedures before handling reagents and samples, always wearing the required PPE, handling implements with care inside the laminar flow cabinet, and developing each step of the process in separate spaces for exclusive use, such as a space to mix reagents, another to add the sample to be evaluated and another to carry out the amplification process.

The NTC amplification could be caused by contamination but also by primer dimerization. For this reason, another critical step is the primer design and identifying the optimal conditions to prevent primer dimerization. It is critical in the primer design process to find the primer set with thermodynamic parameters that reduce the likelihood of secondary structures forming, which is measured with free energy, so a thermodynamic evaluation of the primer set chosen is required. In this protocol, the PrimerDimer tool¹² was employed for the thermodynamic evaluation; with the Multiplex examination option, each primer is screened against all other primers within the pool for potential dimer formations, and the Dimer Framework Report option reports the framework of the most stable dimer of each primer pair. This information is very helpful in selecting a good set of primers.

Furthermore, identifying the appropriate reagent concentration such as MgSO₄, dNTPs, primers, Bst 3.0 enzyme, and amplification parameters such as T_m and incubation time is critical to preventing primer dimerization during the amplification process. Besides, it is possible to add compounds that reduce the formation of primer dimers during amplification, for example, bovine serum albumin, dimethyl sulfoxide (DMSO)¹⁶, and guanidinium chloride¹⁷.

Another critical aspect of the primer design process is determining the specificity of each primer. For this protocol, this evaluation was made with BLAST. However, it is also important to evaluate the specificity of the primers amplification process using another virus that is phylogenetically related but not related to SARS-CoV-2.

On the other hand, the sensitivity and the detection limit (minimum number of detectable genomes) were not determined during the standardization of the technique. In the first place, the samples delivered by University Hospital Fundación Valle del Lili were not quantified to determine the viral copies number due to internal restrictions derived from the sanitary contingency that was in place at that time in Colombia (2020). The estimate of the concentration of genetic material was approximated with the Cq of the RT-qPCR since the lower the Cq, the higher the viral copies number, while the higher the Cq, the lower the viral copies number. The lowest Cq tested was 14.81, and the highest was 38.93, where no amplification products appear. Additionally, in 2020 the RT-qPCR test approved for use in Colombia was qualitative and not quantitative, that is to say, only determined presence or absence according to Cq. A result with Cq ≥ 40 was negative, but a Cq < 40 was positive. The protocols used in Colombia for the detection of COVID-19 were the Berlin protocol1, the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR diagnostic Panel2, and particularly in University Hospital Fundación Valle del Lili, the Allplex 2019-nCoV Assay3 was used. Unfortunately, we do not have any more samples approved by the ethics committee supplied by University Hospital Fundación Valle del Lili to continue with these experiments.

Additionally, the availability of reagents is a significant barrier to the timely diagnosis of new cases in a global public health emergency the one caused by SARS-CoV-2. As a result, this protocol was created to avoid the use of imported and costly commercial kits and reagents, and the preparation of the amplification buffers is detailed in this protocol. Furthermore, some dyes that could be used in colorimetry assays were evaluated, as well as some considerations for their use (Table 7).

This diagnostic method has some limitations because it does not allow for the quantification of the viral genome in the sample. In addition, color detection is a subjective measure because it depends on the visual capacity of the person performing the protocol. However, because it does not require specialized equipment or personnel trained in molecular biology, this protocol is adaptable to the detection of different pathogens and could be easily implemented once it has been standardized. Thus, its application could be extended to other samples, such as environmental^18,19,20 and health centers, to carry out timely epidemiological surveillance.