RAPID DIAGNOSTIC AGENT FOR THE DETECTION OF THE SARS-COV-2 VIRUS.

Abstract

The present invention relates to a method for synthesizing α-Fe2O3 / Fe3O4 nanoparticles having Bst3 polymerase immobilized on their surface, the method being characterized in that it comprises the following steps: 1) synthesis of α-Fe2O3 / Fe3O4 nanoparticles; a) dissolving chitosan in an acid solution to obtain a chitosan solution; b) adding a glutaraldehyde solution to the chitosan solution; c) stirring to form a chitosan hydrogel; d) mineralizing magnetic nanoparticles in the chitosan hydrogel; e) resuspending the chitosan hydrogel in distilled water in an FeCl3 solution at least once to form a CS-Fe complex; f) wetting the CS-Fe complex with a NaOH solution to obtain a black magnetite / chitosan compound; g) degrade the magnetite / chitosan compound in acetic acid and an H2O2 solution to obtain magnetic chitosan nanoparticles;h) Recover the α-Fe2O3 / Fe3O4 nanoparticles by centrifugation; i) Functionalize the α-Fe2O3 / FeO4 nanoparticles; 2) Immobilization of the Bst3 polymerase; a) Take a sample of 0.5 to 1.5 g of functionalized magnetic nanoparticles; b) Mix the functionalized magnetic nanoparticles with glutaraldehyde to a final concentration of 2 to 5%; c) Wash the functionalized magnetic nanoparticles with sodium phosphate buffer and resuspend them in the same buffer; d) Immobilize the Bst3 polymerase by mixing 7,000 to 15,000 units of enzyme with the nanoparticles suspended in sodium phosphate buffer; e) Mix the nanoparticles suspended in sodium phosphate buffer with the enzyme by stirring for 18 to 30 hours; f) separate the nanoparticles suspended in sodium phosphate buffer mixed with the enzyme using a magnet and resuspend them in a sodium phosphate buffer containing 20 to 30% sterile glycerol.;

Description

RAPID DIAGNOSTIC AGENT FOR THE DETECTION OF THE SARS-CoV-2 VIRUS FIELD OF INVENTION The present invention relates to the detection of the SARS-CoV-2 virus and more particularly to a rapid diagnostic agent for the detection of the SARS-CoV-2 virus. BACKGROUND OF THE INVENTION The SARS-CoV-2 virus was first detected in December 2019 in Wuhan, China. Due to the virus's high rate of transmission, the World Health Organization (WHO) declared a pandemic on March 11, 2020. The pandemic has resulted in the deaths of thousands of people worldwide, and the economy has been severely impacted due to social distancing and isolation measures implemented to try to control the spread of the virus. Currently, there are several ways to detect the SARS-CoV-2 virus, such as the Polymerase Chain Reaction (PCR) test, which uses a nasal or oral swab to detect a portion of the virus's genetic material. However, this type of test is not entirely reliable. If there is insufficient viral load, the test may not detect the virus, as there is a time lag between exposure and the moment the viral load is detectable enough for a positive result. Furthermore, a negative test result does not rule out the possibility of rapid infection. If a person is tested too soon after exposure, they may receive a negative result even if they later become infected.Another way to detect the virus is through blood tests and serological or antibody tests, which are performed using a blood sample to detect the antibodies the body produces to fight the virus. These tests are best suited to determine if a person has been exposed to the virus in the past, although they are not recommended to indicate if a person is currently infected. The antigen test, on the other hand, is performed using a nasal swab and detects the protein that coats SARS-CoV-2. However, this test is less sensitive than the PCR test, and there is a possibility of obtaining false negatives in asymptomatic individuals and in people with a few or many days of infection. Loop-mediated isothermal amplification (LAMP) is a single-tube technique for DNA amplification that uses four to six primers to amplify the target sequence by strand displacement with the help of the Bst DNA polymerase enzyme, which operates at a constant temperature of 60 to 65 °C. The use of six primers has been shown to increase the amplification rate and specificity of the LAMP assay. It has the advantage of exhibiting greater stability against inhibitors present in biological samples, which would normally affect the performance of conventional PCR, thus saving time and reducing sample processing costs. Currently, several types of tests and methods exist for detecting the SARS-CoV-2 virus. For example, document CN111321249A describes a kit and method for detecting SARS-CoV-2 using the LAMP technique, comprising a set of primers. Specifically, this document uses primers in conjunction with a reaction buffer, Bacillus stearothermophilus (Bst) DNA polymerase, and reverse transcriptase for SARS-CoV-2 detection. However, this document does not indicate high stability in the test or at any stage of the detection method. Document CN102851405A refers to a kit for detecting H6N2 avian influenza virus DNA using a LAMP technique with gold nanoparticles. The document describes gold nanoparticles labeled with molecular probes capable of identifying a specific sequence of the H6N2 avian influenza virus, and a loop-mediated isothermal nucleic acid amplification system capable of amplifying H6N2 avian influenza virus DNA in a sample for detection, or capable of amplifying H6N2 avian influenza virus DNA in the sample for detection after reverse transcription. Although this document describes the use of gold nanoparticles, it does not describe their use in coupling or immobilizing a polymerase enzyme to said gold nanoparticles, nor does it describe how they provide improved test stability. Finally, the scientific article by Zhu, X., et al., (2020), addresses the diagnosis of COVID-19 using the RT-LAMP (Reverse transcription loop-mediated isothermal amplification) technique coupled with nanoparticle-based biosensors (NBS). This document describes two sets of designed LAMP primers, which were simultaneously amplified and detected in a single-step, single-tube reaction, and the detection results were interpreted by nanoparticle-based biosensors. However, this document does not indicate that the use of nanoparticle-based biosensors provides better test stability. As a result, efforts have been made to overcome the drawbacks of currently used SARS-CoV-2 tests and detection kits by developing a rapid diagnostic agent for SARS-CoV-2 detection that utilizes nanoparticles to achieve high stability, thus increasing its reliability. It also allows for the qualitative detection of SARS-CoV-2 in samples with low viral concentrations from COVID-19 patients. Furthermore, it is a rapid, sensitive, and easy-to-interpret agent. This rapid diagnostic agent for SARS-CoV-2 detection does not require refrigeration, facilitating its easy distribution, even in remote communities, and its low production cost makes it accessible to most of the population. OBJECTS OF THE INVENTION Taking into account the shortcomings of the prior art, it is an object of the present invention to provide a rapid diagnostic agent for the detection of the SARS-CoV-2 virus that utilizes nanoparticles to achieve high stability, thereby increasing its reliability. Another object of the present invention is to provide a rapid diagnostic agent for the detection of the SARS-CoV-2 virus that allows qualitative detection of the presence of SARS-CoV-2 in samples with low viral concentrations from patients with COVID-19. It is yet another object of the present invention to provide a rapid diagnostic agent for the detection of the SARS-CoV-2 virus that is fast, sensitive and easy to interpret. Another object of the present invention is to provide a rapid diagnostic agent for the detection of the SARS-CoV-2 virus that does not require refrigeration, thus allowing for its easy distribution, even in remote communities. It is yet another object of the present invention to provide a rapid diagnostic agent for the detection of the SARS-CoV-2 virus that has a low production cost, making it accessible to most of the population. Finally, it is an object of the present invention to provide a rapid diagnostic agent for the detection of the SARS-CoV-2 virus that does not require expensive equipment, such as thermocyclers, hard-to-access materials, or trained personnel for the interpretation of the result. These and other objectives are achieved through the rapid diagnostic agent for the detection of the SARS-CoV-2 virus in accordance with the present invention. BRIEF DESCRIPTION OF THE INVENTION, A rapid diagnostic agent for the detection of the SARS-CoV-2 virus has been invented, characterized in that it comprises a-Fe2O3 / Fe3O4 nanoparticles with Bst3 polymerase immobilized on the surface of said a-Fe2O3 / Fe3O4 nanoparticles. Another aspect of the present invention relates to the synthesis of α-Fe2O3 / Fe3O4 nanoparticles comprising: a) dissolving chitosan in an acetic acid solution to obtain a chitosan solution; b) adding a glutaraldehyde solution to the chitosan solution; c) stirring to form a chitosan hydrogel; d) mineralizing magnetite nanoparticles in the chitosan hydrogel; e) resuspending the chitosan hydrogel in distilled water in an FeCl3 solution at least once to form a CS-Fe complex; f) wetting the CS-Fe complex with a NaOH solution to obtain a black magnetite / chitosan compound; g) degrading the magnetite / chitosan compound in acetic acid and an H2O2 solution to obtain magnetic chitosan nanoparticles; h) recover the a-Fe2O3 / Fe3O4 nanoparticles by centrifugation; and i) functionalize the aFe2O3 / Fe3O4 nanoparticles. Another aspect of the present invention relates to an aFe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus with the Bst3 polymerase immobilized on its surface. Another aspect of the present invention is a rapid diagnostic kit for the detection of the SARS-CoV-2 virus, comprising a LAMP-PCR test for diagnosing the presence of the SARS-CoV-2 virus in patients with COVID-19, which uses a rapid diagnostic agent with a-Fe2O3 / Fe3O4 nanoparticles, salts, stabilizers, dNTPs, primers, dye and ORFlab sequences of the SARS-CoV-2 virus. An additional aspect of the present invention is a rapid diagnostic method for the detection of the SARS-CoV-2 virus, comprising: a) taking a sample of the patient's exudate; b) denaturing the patient's exudate sample to obtain SARS-CoV-2 virus RNA; c) detecting the presence of the SARS-CoV-2 virus using SARS-CoV-2 virus-specific synthetic sequences from ORFlab; and d) determining whether the test result is negative or positive. BRIEF DESCRIPTION OF THE DRAWINGS The novel aspects considered characteristic of the present invention will be set forth in detail in the appended claims. However, some embodiments, features, and some objects and advantages thereof will be better understood in the detailed description when read in conjunction with the accompanying drawings, in which: Figure 1 shows the X-ray diffraction results of the a-Fe2O3 / Fe3O4 nanoparticles according to the present invention. Figure 2 shows the Fourier transform infrared (FTIR) spectroscopy analysis of α-Fe2O3 / Fe3O4 nanoparticles, chitosan and chitosan with α-Fe2O3 / Fe3O4 nanoparticles according to the present invention. Figure 3 shows the colorimetric results of the samples and their respective controls in accordance with the present invention. Figure 4 shows the results obtained from the samples after being exposed to UV-Vis light where fluorescence is produced in accordance with the present invention. Figure 5 shows a graph of the evaluation of the limits of detection and exploration of the quantitative method of viral loads by spectrophotometry in accordance with the present invention. Figure 6 shows the results of the samples obtained from a LAMPPCR test with α-Fe2O3 / Fe3O4 nanoparticles in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION The present invention offers certain advantages over the prior art, including the use of nanoparticles in the rapid diagnostic agent for detecting the SARS-CoV-2 virus, which achieves high stability and thus increases its reliability. It allows for the qualitative detection of SARS-CoV-2 in samples with low viral concentrations from COVID-19 patients. The rapid, sensitive, and easy-to-interpret diagnostic test for SARS-CoV-2 does not require refrigeration, facilitating its easy distribution, even in remote communities. Furthermore, it has a low production cost, making it accessible to most of the population, and does not require expensive equipment, hard-to-obtain materials, or trained personnel for interpretation. Therefore, one aspect of the present invention is a rapid diagnostic agent for the detection of the SARS-CoV-2 virus, comprising a-Fe2O3 / Fe3O4 nanoparticles with Bst3 polymerase immobilized on the surface of said a-Fe2O3 / Fe3O4 nanoparticles. In a preferred embodiment of the present invention, the diagnostic agent is an agent that can be used in a LAMP-PCR test. Preferably, the diagnostic agent further comprises the following components: KCl, (NH4)2SO4, betaine, MgCl2, Tween®, dNTPs, and the oligonucleotides F1-1 / B1-1, F1-3 / B1-3, and F1-2 / B1-2. More preferably, the component concentrations are: KCl 45 to 55 mM, (NH4)2SO45 to 10 mM, betaine 0.2 to 0.6 M, MgCl24 to 8 mM, Tween® 0.1%, dNTPs 0.5 to 2 mM, Fl-l / Bl-1 1.4 to 2 mM, F1-3 / B1-3, 0.1 to 0.4 mM and F1-2 / B1-2 to 0.4 mM. In a preferred embodiment, the a-Fe2O3 / Fe3O4 nanoparticles have a size of between 10 and 30 nm. Another aspect of the present invention relates to the synthesis of α-Fe2O3 / Fe3O4 nanoparticles comprising: a) dissolving chitosan in an acetic acid solution to obtain a chitosan solution; b) adding a glutaraldehyde solution to the chitosan solution; c) stirring to form a chitosan hydrogel; d) mineralizing magnetite nanoparticles in the chitosan hydrogel; e) resuspending the chitosan hydrogel in distilled water in an FeCl3 solution at least once to form a CS-Fe complex; f) wetting the CS-Fe complex with a NaOH solution to obtain a black magnetite / chitosan compound; g) degrading the magnetite / chitosan compound in acetic acid and an H2O2 solution to obtain magnetic chitosan nanoparticles; h) recover the a-Fe2O3 / Fe3O4 nanoparticles by centrifugation; and i) functionalize the aFe2O3 / Fe3O4 nanoparticles. Preferably, chitosan is present at a concentration of 0.002 to 0.006 M in the acetic acid solution. Preferably, the acetic acid solution is present at a concentration of 1 to 5% (v / v). Preferably, the glutaraldehyde solution is present at a concentration of 20 to 30% (v / v). Preferably, 45 to 55 mL of the chitosan solution are measured out. Preferably, 0.1 to 0.5 mL of glutaraldehyde solution is added to the previously measured chitosan solution. Preferably, the glutaraldehyde solution is added to the chitosan solution under constant stirring at 600 rpm. Preferably, constant stirring is maintained for 22 to 26 hours. Preferably, two solutions were prepared for the mineralization of the magnetite nanoparticles. Preferably, the first solution is prepared with FeCl3 at a concentration of 0.05 to 0.25 mol / L using FeCl36H2O. Preferably, the second solution is prepared with FeCl2 at a concentration of 0.05 to 0.0.9 mol / L using FeCl₂-4H₂O. Preferably, the pH of both solutions is adjusted to 1. In a preferred embodiment, the chitosan hydrogel is soaked in the FeCl₂ solution for 20 to 40 minutes and washed with deionized water. In a preferred embodiment, the CS-Fe complex is soaked in a NaOH solution with a concentration of 1 to 1.5 M. Preferably, the magnetite / chitosan composite is degraded in 3% (v / v) acetic acid and a 5% (v / v) H₂O₂ solution. Preferably, after the magnetic chitosan nanoparticles are recovered by centrifugation, they are washed with distilled water until a neutral pH is reached. It should be noted that the nanoparticle synthesis was achieved using methods that did not require subsequent synthesis processes and were obtained at low temperatures, resulting in low production costs and energy savings. In a preferred embodiment of the present invention, the functionalization of the α-Fe2O3 / Fe3O4 nanoparticle comprises: a) resuspending the chitosan magnetic nanoparticles in distilled water in a methanol solution to obtain a chitosan-methanol solution; b) ultrasonically mixing the chitosan-methanol solution; c) adding NH3H2O ​​to the chitosan-methanol solution; d) ultrasonically mixing the chitosan-methanol solution again; e) adding 3-APTES to the solution; f) heating and stirring the chitosan-methanol solution; g) separating the chitosan magnetic nanoparticles by magnetic decantation; h) obtaining functionalized magnetic nanoparticles; and i) immobilizing the Bst3 polymerase on the functionalized magnetic nanoparticles. In a preferred embodiment of the present invention, the amount of nanoparticles functionalized is 0.5 to 3 grams. Preferably, the methanol solution has a concentration of 10 to 14 M. Preferably, the non-methanol chitosan solution is ultrasonically mixed for 20 to 40 minutes. Preferably, 1 to 6 mL of NH3H2O ​​is added to the chitosan-methanol solution. Preferably, the chitosan-methanol solution is again ultrasonically mixed. Preferably, 1 to 5 mL of the 3-APTES solution is added to the chitosan-methanol solution. Preferably, the chitosan-methanol solution with 3-APTES is heated to a temperature of 40 to 70°C. Preferably, the magnetic nanoparticles separated by magnetic decantation are washed with ethanol and water until a neutral pH is reached. In another embodiment of the present invention, a 0.5 to 1.5 g sample of functionalized magnetic nanoparticles is taken to immobilize the Bst3 polymerase on the magnetic nanoparticles. Preferably, the functionalized magnetic nanoparticles are mixed with glutaraldehyde to a final concentration of 2 to 5%. Preferably, the functionalized magnetic nanoparticles are stirred at room temperature. More preferably, stirring is performed at a speed of 80 to 150 rpm for 2 to 6 hours. Preferably, the functionalized magnetic nanoparticles are separated until the mixture appears clear, and decanting is performed. Preferably, the functionalized magnetic nanoparticles are washed with sodium phosphate buffer and resuspended in the same buffer. Preferably, the Bst3 polymerase is immobilized by mixing 7,000 to 15,000 enzyme units with nanoparticles suspended in sodium phosphate buffer.Preferably, the amount of nanoparticles suspended in sodium phosphate buffer mixed is 0.05 to 0.9 mg. Preferably, the sodium phosphate buffer has a neutral pH. Preferably, the nanoparticles suspended in sodium phosphate buffer mixed with the enzyme are stirred for 18 to 30 hours. In a preferred embodiment, the nanoparticles suspended in sodium phosphate buffer mixed with the enzyme are separated using a magnet and resuspended in a sodium phosphate buffer containing 20 to 30% sterile glycerol. Another aspect of the present invention relates to an aFe2O3 / Fe3O4 nanoparticle for detecting the SARS-CoV-2 virus with the Bst3 polymerase immobilized on its surface. It should be noted that the functionalization process allows the addition of functional groups belonging to 3-APTES to the surface of the nanoparticles where the enzyme is anchored. A further aspect of the present invention is a rapid diagnostic kit for the detection of the SARS-CoV-2 virus, comprising a LAMP-PCR test for diagnosing the presence of the SARS-CoV-2 virus in patients with COVID-19, which uses a rapid diagnostic agent with a-Fe2O3 / Fe3O4 nanoparticles, salts, stabilizers, dNTPs, primers, dye and ORFlab sequences of the SARS-CoV-2 virus. In a preferred embodiment of the present invention, the rapid diagnostic agent with α-Fe2O3 / Fe3O4 nanoparticles is coupled to the LAMP-PCR assay to provide greater stability to the reaction components. This allows the rapid diagnostic agent to immobilize the Bst3 polymerase enzyme using the α-Fe2O3 / Fe3O4 nanoparticles, enabling the accurate and rapid detection of the SARS-CoV-2 virus, even at low viral loads. It should be noted that the characteristics of the loop-mediated isothermal amplification (LAMP) technique, using the rapid diagnostic agent with α-Fe2O3 / Fe3O4 nanoparticles, allow the amplification process to be carried out using a water bath without the need for specialized equipment. Preferably, the salts used are selected from NH4Cl and (NH4)2SO4. Preferably, the stabilizer used is BSA. Preferably, 5 to 20 mM dNTPs are used. In another preferred embodiment, the dye is selected from fluorescent dyes. Preferably, the fluorescent dye is selected from SYBR Gold, SYBR Green, red gel, or a mixture thereof. Preferably, the set of primers used comprises at least one sequence-directed primer Fl-3, one sequence-directed primer Fl-2, one sequence-directed primer Fl-1, one sequence-directed primer Bl-1, one sequence-directed primer Bl-2, and one sequence-directed primer Bl-3. More preferably, primer Fl-3 corresponds to sequence SEQ ID NO: 1. More preferably, primer Fl-2 corresponds to sequence SEQ ID NO: 2. More preferably, primer Fl-1 corresponds to sequence SEQ ID NO: 3. More preferably, primer Bl-1 corresponds to sequence SEQ ID NO: 4. More preferably, primer Bl-2 corresponds to sequence SEQ ID NO: 5. More preferably, primer Bl-3 corresponds to sequence SEQ ID NO: 6. Preferably, the ORFlab sequence of the SARS-CoV-2 virus has a length of 921 base pairs. More preferably, the ORFlab sequence of the SARS-CoV-2 virus corresponds to the sequence SEQ ID NO: 7. An additional aspect of the present invention is a rapid diagnostic method for the detection of the SARS-CoV-2 virus, comprising: a) taking a sample of the patient's exudate; b) denaturing the patient's exudate sample to obtain SARS-CoV-2 virus RNA; c) detecting the presence of the SARS-CoV-2 virus using SARS-CoV-2 virus-specific synthetic sequences from ORFlab; and d) determining whether the test result is negative or positive. Preferably, the exudate is a pharyngeal swab. Preferably, the exudate is collected using a swab or tongue depressor. Preferably, the presence of the SARS-CoV-2 virus is detected using a pH-based colorimetric indicator. More preferably, the colorimetric indicator is selected from hydroxynaphthol blue, phenol red, or a mixture of both. Preferably, the colorimetric indicator changes to purple if the test result is negative and to blue if the test result is positive. It should be noted that in the presence of the SARS-CoV-2 virus, the pH-based colorimetric indicator amplifies the DNA strand for each nucleotide added to the sequence, releasing a hydrogen atom. This acidifies the medium, resulting in a color change. The present invention will be better understood from the following examples, which are presented for illustrative purposes only to allow a full understanding of the preferred embodiments of the present invention, without implying that there are no other unillustrated embodiments that can be put into practice based on the detailed description above. EXAMPLE 1 An experiment was conducted to synthesize and functionalize a-Fe2O3 / Fe3O4 nanoparticles in accordance with the present invention. For this experiment, the nanoparticles were obtained as follows: 1.5 g of chitosan was dissolved in a 2% (v / v) acetic acid solution and stirred constantly until completely dissolved. Then, 0.2 mL of 25% glutaraldehyde solution was added to 50 mL of chitosan solution under vigorous stirring. The solution was allowed to stand for 24 h until the chitosan hydrogel was fully formed. The mineralization of magnetite nanoparticles in chitosan hydrogel was then carried out using two solutions: a) 0.15 mol / L FeCl3 using FeCl3·6H2O and b) 0.075 mol / L FeCl2 using FeCl2·4H2O. The pH of both solutions was adjusted to approximately 1. The chitosan hydrogel was then soaked in the FeCl3 solution for 30 minutes. Afterward, the hydrogel was washed with deionized water and soaked in the FeCl2 solution for 30 minutes, followed by washing with deionized water. This cycle was repeated four times to form the CS-Fe complex (chitosan-magnetic particles). Finally, the CS-Fe complex was soaked in a 1.25 mol / L NaOH solution for 12 h, yielding a black magnetite / chitosan compound. Finally, to obtain the magnetic nanoparticles, the magnetite / chitosan compound was degraded in 3% (v / v) acetic acid and a 5% (v / v) H2O2 solution.The iron nanoparticles were recovered by centrifugation and washed with distilled water until a neutral pH was reached. EXAMPLE 2 A test was performed to determine the characteristics of the aFe2O3 / Fe3O4 nanoparticle obtained in the previous example in accordance with the present invention. To determine the crystalline structure of the iron nanoparticles, they were analyzed using powder X-ray diffraction (XRD). However, given their nature, a second analysis was required to determine the oxidation state of the iron. For this, X-ray photoelectron spectroscopy (XPS) was used. Additionally, Fourier transform infrared spectroscopy (FTIR) was used to determine the functional groups present in the nanoparticles. Finally, scanning electron microscopy (SEM) was used to study the microstructure of the nanoparticles. Figure 1 shows the characterization results of the α-Fe₂O₃ / Fe₃O₄ nanoparticles obtained in the previous example in accordance with the present invention. Figure 1 shows the X-ray diffraction (XRD). This study allows us to determine the crystalline structure of the material and thus whether the nanoparticles have the desired phase. Figure 2 shows the Fourier transform infrared spectroscopy (FTIR) analysis of α-Fe2O3 / Fe3O4 nanoparticles, chitosan, and chitosan with 15 α-Fe2O3 / Fe3O4 nanoparticles. This study investigated the differences between nanoparticles obtained with chitosan versus nanoparticles obtained without using chitosan. EXAMPLE 3 A trial was conducted to manufacture a rapid diagnostic agent for the detection of the SARS-CoV-2 virus in accordance with the present invention. For this assay, the Bst3 polymerase was first immobilized as follows: 0.8 g of glutaraldehyde-functionalized nanoparticles were mixed to a final concentration of 3% and stirred at 100 rpm at room temperature (25 °C) for 4 hours. The nanoparticles were then separated using a magnet until the mixture became clear and were removed by decantation. They were subsequently washed three times with 50 mM sodium phosphate buffer, pH 7.2, and resuspended in the same buffer. To immobilize the Bst enzyme, 10,000 enzyme units were mixed with 0.1 mg of nanoparticles suspended in sodium phosphate buffer, pH 7.2, and stirred at 100 rpm for 24 hours in the dark at 25 °C. They were separated with a magnet and resuspended in 0.1 mL of sodium phosphate buffer containing 25% sterile glycerol.Reaction 30 was established at a temperature of 55 °C for time ranges of 35-50 minutes depending on the viral standard concentrations, which were 1-25 ng. The elements contained in the reaction were: KCl 50 mM, (NH4)2SO4 10 mM, Betaine 0.4 M, MgCl 26 mM, Tween 0.1%, dNTPs 1 mM, and F1 / B1-1 1.6 mM, F1-3 / B1-3 0.2 mM, F1-2 / B1-2, and oligonucleotides. EXAMPLE 4 An assay was performed to obtain a protocol for a LAMP-PCR test without immobilizing the Bst3 polymerase enzyme in accordance with the present invention. This trial sought to establish a functional baseline protocol and designed a protocol that included the standard components of the LAMP-PCR test, including salts, stabilizers, dNTPs, primers, dye and synthetic DNA standards, with ORFlab sequences of the SARS-CoV-2 virus. For this assay, the mixture contained in each reaction of the kit consisted of: 50 mM KCl, 10 mM (NH4)2SO4, 0.4 M Betaine, 26 mM MgCl, 0.1% Tween, 1 mM dNTPs, 1.6 mM F1-1 / B1-1, 0.2 mM F1-3 / B1-3, F1-2 / B1-2, and oligonucleotides. The main variant used in this assay was the Bst3 polymerase enzyme, which was not immobilized with nanoparticles. DNA amplification required repeated trials under different conditions and concentrations to be optimized and standardized according to a protocol. The reaction was carried out at 55 °C for 35–50 minutes, depending on the viral standard concentrations, which ranged from 1 to 25 ng. It should be noted that, once the protocol was established, a change from purple to blue was achieved in the hydroxynaphthol blue pH colorimetric indicator.It is important to mention that the color change signifies DNA amplification, because the dye under an alkaline pH acquires a purple hue. As amplification occurs, for each nucleotide that the polymerase adds to the DNA strand, an H+ is released, resulting in the acidification of the medium, which produces a color change from purple to blue. Figure 3 shows the colorimetric results of the samples and their respective controls. Item 1 shows a sample obtained with free Bst3 polymerase (i.e., without immobilization) under the following conditions: presence of the ORFlab sequence of the SARS-CoV-2 virus subjected to PCR-LAMP and isothermal PCR testing. Items 2 and 3 show a sample obtained with free Bst3 polymerase (i.e., without immobilization) under the following conditions: presence of the ORFlab sequence of the SARS-CoV-2 virus subjected to PCR-LAMP and without being subjected to isothermal PCR. Item 4 shows a sample obtained with free Bst3 polymerase (i.e., without immobilization) under the following conditions: presence of the ORFlab sequence of the SARS-CoV-2 virus subjected to PCR-LAMP and with corresponding B DNA.In section 5, a sample obtained with free Bst3 polymerase (i.e., without immobilization) is shown under the following conditions: presence of the ORFlab sequence of the SARS-CoV-2 virus subjected to PCR-LAMP and the test subjected to isothermal PCR. In section 6, a sample obtained with free Bst3 polymerase (i.e., without immobilization) is shown under the following conditions: presence of the ORFlab sequence of the SARS-CoV-2 virus subjected to PCR-LAMP and without a DNA sample. Additionally, in order to demonstrate the presence of DNA at the end of the process LAMP-PCR, with free Bst3 polymerase, was stained with the intercalating agent Sybr Gold; This agent binds to the double-stranded molecules of DNA, and when exposed to UV-Vis light it produces fluorescence. Figure 4 shows the samples obtained after exposure to UV-Vis light, where fluorescence occurs. Item 1 shows a sample containing the ORFlab sequence of the SARS-CoV-2 virus, subjected to LAMP PCR and isothermal PCR. Items 2 and 3 show a sample containing the ORFlab sequence of the SARS-CoV-2 virus, subjected to LAMP PCR but not to isothermal PCR. Item 4 shows a sample containing the ORFlab sequence of the SARS-CoV-2 virus, subjected to LAMP PCR and corresponding B DNA, and subjected to isothermal PCR. Item 5 shows a sample containing the ORFlab sequence of the SARS-CoV-2 virus, subjected to LAMP PCR and corresponding B DNA, and subjected to isothermal PCR. In section 6, a sample is observed with the presence of the ORFlab sequence of the SARS-CoV-2 virus, subjected to LAMP PCR and without a DNA sample. Based on these results, it was concluded that the protocol designed as the basis for the kit's development functions adequately, sensitively, and selectively for the detection of SARS-CoV-2 when using the polymerase in its free form. Subsequently, the same tests were performed, standardized, and optimized using the polymerase enzyme immobilized on nanoparticles. EXAMPLE 4 A test was performed to determine the sensitivity of the PCR-LAMP test in accordance with the present invention. For this assay, the sensitivity of the PCR-LAMP test described in the previous example was determined. A detection curve was established with standard concentrations ranging from 1-25 ng / L, in which the color change was observed at concentrations of 5 ng / L. Figure 5 shows a graph of the detection and exploration limits of the quantitative viral load method using spectrophotometry. The graph can be interpreted quantitatively through a spectrophotometric scan from 400 to 700 nm, by observing the color change at different concentrations and the resulting decrease in the absorption spectrum. EXAMPLE 5 An assay was performed to activate the nanoparticles and immobilize the components of the diagnostic agent with a-Fe2O3 / Fe3O4 nanoparticles in accordance with the present invention. For this assay, 1 g of chitosan / iron nanoparticles were resuspended in distilled water in 30 mL of methanol solution and mixed under ultrasonication for 30 minutes. Then, 3 mL of NH3H2O ​​(25 wt%) were added to the solution, and it was ultrasonographed for an additional 10 minutes. Subsequently, 2 mL of 3-APTES were added dropwise under vigorous stirring. The solution was heated to 50 °C and stirred continuously for 8 h using recirculation. The resulting 3-APTES-modified chitosan / iron nanoparticles were separated by magnetic separation and washed with ethanol and water, respectively, until a neutral pH was reached. To immobilize the Bst3 polymerase, 0.8 g of functionalized nanoparticles were mixed with glutaraldehyde to a final concentration of 3% and stirred at 100 rpm at room temperature (25 °C) for 4 hours.The nanoparticles were separated using a magnet until the mixture became clear and was then removed by decantation. Subsequently, the nanoparticles were washed three times with 50 mM sodium phosphate buffer, pH 7.2, and resuspended in the same buffer. To immobilize the Bst3 polymerase, 10,000 enzyme units were mixed with 0.1 mg of nanoparticles suspended in sodium phosphate buffer, pH 7.2, and stirred at 100 rpm for 24 hours in the dark at 25 °C. The nanoparticles were then separated using a magnet and resuspended in 0.1 mL of sterile sodium phosphate buffer containing 25% glycerol. EXAMPLE 6 A test was performed to standardize the diagnostic agent with a-Fe2O3 / Fe3O4 nanoparticles in accordance with the present invention. For this assay, previously conducted tests with the commercially available, non-immobilized Bst3 polymerase enzyme were repeated, with the difference that in this stage it was replaced with Bst3 polymerase immobilized on α-Fe2O3 / Fe3O4 nanoparticles. The protocol was adapted and optimized for better performance with this new variable. In this experimental phase, it was observed that the immobilized polymerase produced an effect very similar to its non-immobilized counterpart. Figure 5 shows the colorimetric results of samples obtained with the Bst3 polymerase enzyme immobilized on α-Fe2O3 / Fe3O4 nanoparticles. The colorimetric results obtained with the polymerase enzyme immobilized on the α-Fe2O3 / Fe3O4 nanoparticles are comparable to those obtained with the enzyme without immobilization. However, samples containing this duplex do not require special handling such as refrigeration or specific storage conditions, which reduces overall costs. Figure 3 shows the samples obtained with the Bst3 polymerase enzyme immobilized on the α-Fe2O3 / Fe3O4 nanoparticles. In section 1, a sample containing the ORFlab sequence of the SARS-CoV-2 virus, subjected to LAMP PCR and isothermal PCR, is shown. In sections 2 and 3, a sample with the presence of the ORFlab sequence of the SARS-CoV-2 virus is observed, subjected to LAMP PCR and not subjected to isothermal PCR.Item 4 shows a sample containing the ORFlab sequence of the SARS-CoV-2 virus, subjected to LAMP PCR and corresponding B DNA, and subjected to isothermal PCR. Item 5 shows a sample containing the ORFlab sequence of the SARS-CoV-2 virus, subjected to LAMP PCR and corresponding B DNA, and subjected to isothermal PCR. Item 6 shows a sample containing the ORFlab sequence of the SARS-CoV-2 virus, subjected to LAMP PCR, but without a DNA sample. EXAMPLE 7 An assay was performed to demonstrate the specificity for the detection and amplification only of genetic material corresponding to SARS-CoV-2 in accordance with the present invention. This test was performed in order to demonstrate the specificity for the detection and amplification only of genetic material corresponding to SARS-CoV-2 of the experimental design 15 and the same protocol was performed with genomic DNA of BaciHus subtuis and Fusarium oxysporum, in this way it was possible to corroborate that the amplification would only occur in the presence of the corresponding viral sequence. Figure 6 shows the samples obtained from a LAMP-PCR test using α-Fe₂O₃ / Fe₃O₄ nanoparticles. In section A), the samples with the SARS-CoV-2 virus sequence 20 ORFlab are shown, exhibiting a significant color change from purple to blue due to amplification of the segment. In section B), the samples with BaciHus subtiUs DNA are shown, which do not exhibit a color change because there is no amplification. In section C), the samples with Fusarium oxysporum DNA are shown. As can be seen in this test, the samples obtained by means of a 25 LAMP-PCR test have greater stability of their components and their sensitivity to detect positive results is greater than in a LAMP-PCR test in which the Bst3 polymerase is not immobilized. In accordance with the foregoing, it will be observed that the rapid diagnostic agent for the detection of the SARS-CoV-2 virus has been designed to achieve high stability, thereby increasing its reliability, and it will be evident to any expert in the field that the modalities of the rapid diagnostic agent for the detection of the SARS-CoV-2 virus as described above and illustrated in the accompanying drawings are merely illustrative and not limiting to the present invention, since numerous significant changes in its details are possible without departing from the scope of the invention. Therefore, the present invention shall not be considered restricted except as required by prior art and within the scope of the appended claims.

Claims

1. A rapid diagnostic agent for the detection of the SARS-CoV-2 virus, 5 characterized in that it comprises a-Fe2O3 / Fe3O4 nanoparticles with Bst3 polymerase immobilized on the surface of said a-Fe2O3 / Fe3O4 nanoparticles.

2. The rapid diagnostic agent for the detection of the SARS-CoV-2 virus according to claim 1, further characterized in that the diagnostic agent is an agent that can be used in a LAMP-PCR test. 10 3. The rapid diagnostic agent for the detection of the SARS-CoV-2 virus according to claim 1, further characterized in that the diagnostic agent comprises the components: KCI, (NH4)2SO4, betaine, MgCI2, Tween®, dNTPs, and the oligonucleotides Fl-l / Bl-1, F1-3 / B1-3, F1-2 / B1-2.

4. The rapid diagnostic agent for the detection of the SARS-CoV-2 virus according to claim 3, further characterized in that the concentrations of the components are: KCl 45 to 55 mM, (NH4)2SO4 5 to 10 mM, betaine 0.2 to 0.6 M, MgCl2 4 to 8 mM, Tween® 0.1%, dNTPs 0.5 to 2 mM, Fl-l / Bl-1 1.4 to 2 mM, F1-3 / B1-3 0.1 to 0.4 mM and F1-2 / B1-2 0.4 mM.

5. The rapid diagnostic agent for the detection of the SARS-CoV-2 virus according to claim 1, further characterized in that the aFe2O3 / Fe3O4 nanoparticles have a size of between 10 and 30 nm.

6. The synthesis of an α-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus, characterized in that it comprises: a) dissolving chitosan in an acetic acid solution to obtain a chitosan solution; b) adding a glutaraldehyde solution to the chitosan solution; c) stirring to form a chitosan hydrogel; d) mineralizing magnetite nanoparticles in the chitosan hydrogel; e) resuspending the chitosan hydrogel in distilled water in an FeCl3 solution at least once to form a CS-Fe complex; f) wetting the CS-Fe complex with a NaOH solution to obtain a black magnetite / chitosan compound; g) degrading the magnetite / chitosan compound in acetic acid and an H2O2 solution to obtain magnetic chitosan nanoparticles; h) recover the a-Fe2O3 / Fe3O4 nanoparticles by centrifugation; and i) functionalize the aFe2O3 / Fe3O4 nanoparticles.

7. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 6, further characterized in that the chitosan is in a concentration of 0.002 to 0.006 M in the acetic acid solution.

8. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 6, further characterized in that the acetic acid solution is at a concentration of 1 to 5% (v / v).

9. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 6, further characterized in that the glutaraldehyde solution is at a concentration of 20 to 30% (v / v).

10. The synthesis of an α-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 6, further characterized in that two solutions are prepared in the mineralization of the magnetite nanoparticles.

11. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 10, further characterized in that the pH of the two solutions is adjusted to 1.

12. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 10, further characterized in that the first 15 solution is prepared with FeCI3a at a concentration of 0.05 to 0.25 mol / L using FeCI36H2O.

13. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 10, further characterized in that the second solution is prepared with FeCI2 at a concentration of 0.05 to 0.09 mol / L using FeCI2'4H2O.

14. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 6, further characterized in that the chitosan hydrogel is soaked in the FeCI2 solution for 20 to 40 minutes and washed with deionized water.

15. The synthesis of an α-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 6, further characterized in that the CS-Fe complex is soaked in a NaOH solution with a concentration of 1 to 1.5 M. 25 16. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 6, further characterized in that the magnetite / chitosan compound degrades into 3% (v / v) acetic acid and a 5% (v / v) H2O2 solution.

17. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 6, further characterized in that after the chitosan magnetic nanoparticles are recovered by centrifugation, they are washed with distilled water until a neutral pH is reached.

18. The synthesis of an α-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 6, further characterized in that the functionalization of the α-Fe2O3 / Fe3O4 nanoparticle comprises: a) resuspending the 35 magnetic chitosan nanoparticles in distilled water in a methanol solution to obtain a chitosan-methanol solution; b) ultrasonically mixing the chitosan-methanol solution; c) adding NH3H2O ​​to the chitosan-methanol solution; d) ultrasonically mixing the chitosan-methanol solution again; e) adding 3-APTES to the solution; f) heating and stirring the chitosan-methanol solution; and g) separating the magnetic chitosan nanoparticles by magnetic decantation; f) obtaining 5 functionalized magnetic nanoparticles; g) immobilize the Bst3 polymerase in the functionalized magnetic nanoparticles.

19. The synthesis of an α-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 18, further characterized in that the methanol solution has a concentration of 10 to 14 M. 10 20. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 18, further characterized in that the chitosan-methanol solution is mixed in ultrasound for 20 to 40 minutes.

21. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 18, further characterized in that 1.15 to 6 ml of NH3H2O ​​are added to the chitosan-methanol solution.

22. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 18, further characterized in that 1 to 5 ml of the 3-APTES solution are added to the chitosan-methanol solution.

23. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus according to claim 18, further characterized in that the chitosan-methanol solution with 3-APTES is heated to a temperature of 40 to 70°C.

24. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus functionalized according to claim 18, further characterized in that the magnetic nanoparticles separated by magnetic decantation are washed with ethanol and water 25 until a neutral pH is reached.

25. The synthesis of an α-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus, functionalized according to claim 18, further characterized in that a sample of 0.5 to 1.5 g of functionalized magnetic nanoparticles is taken to immobilize the Bst3 polymerase on the magnetic nanoparticles. 30 26. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus functionalized according to claim 18, further characterized in that the functionalized magnetic nanoparticles are mixed with glutaraldehyde to a final concentration of 2 to 5%.

27. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus functionalized according to claim 18, further characterized in that the functionalized magnetic nanoparticles are washed with sodium phosphate buffer and resuspended in the same buffer.

28. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus functionalized according to claim 18, further characterized in that the Bst3 polymerase 5 is immobilized by mixing 7,000 to 15,000 enzyme units with nanoparticles suspended in sodium phosphate buffer.

29. The synthesis of an a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus functionalized according to claim 27, further characterized in that the nanoparticles suspended in sodium phosphate buffer mixed with the enzyme are subjected to stirring for 18 to 30 hours.

30. The synthesis of an α-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus, functionalized according to claim 29, further characterized in that the nanoparticles suspended in sodium phosphate buffer mixed with the enzyme are separated with a magnet and resuspended in a sterile sodium phosphate buffer containing 20 to 30% glycerol.

31. An a-Fe2O3 / Fe3O4 nanoparticle for the detection of the SARS-CoV-2 virus with the Bst3 polymerase immobilized on its surface.

32. A rapid diagnostic kit for the detection of the SARS-CoV-2 virus, characterized in that it comprises a LAMP-PCR test for the diagnosis of the presence of the SARS-CoV-2 virus in patients with COVID-19, which uses a rapid diagnostic agent with a-Fe2O3 / Fe3O4 nanoparticles, salts, stabilizers, dNTPs, primers, dye and ORFlab sequences of the SARS-CoV-2 virus.

33. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 32, further characterized in that the rapid diagnostic agent 25 with a-Fe2O3 / Fe3O4 nanoparticles is coupled to the LAMP-PCR test.

34. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 32, further characterized in that the salts used are selected from NH4Cl and (NH4)2SO4.

35. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 32, further characterized in that the stabilizer used is BSA.

36. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 32, further characterized in that 5 to 20 mM of dNTPs are used.

37. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 32, further characterized in that the dye is selected from fluorescent dyes.

38. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 37, further characterized in that the fluorescent dye is selected from SYBR Gold, SYBR green, red gel or a mixture thereof.

39. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 32, further characterized in that the set of primers used comprises at least one sequence-directed Fl-3 primer, one sequence-directed Fl-2 primer, one sequence-directed Fl-1 primer, one sequence-directed Bl-1 primer, one sequence-directed Bl-2 primer, and one sequence-directed Bl-3 primer.

40. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 39, further characterized in that the primer Fl-3 corresponds to the sequence SEQ ID NO:

1.

41. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 39, further characterized in that the primer Fl-2 corresponds to the sequence SEQ ID NO:

2. ψ 42. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus of ti . 20 in accordance with claim 39, further characterized in that the primer Fl-1 corresponds to the sequence SEQ ID NO:

3.

43. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 39, further characterized in that the primer Bl-1 corresponds to the sequence SEQ ID NO: 4.25 44. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 39, further characterized in that the primer Bl-2 corresponds to the sequence SEQ ID NO:

5.

45. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 39, further characterized in that the primer Bl-3 corresponds to the sequence SEQ ID NO:

6.

46. ​​The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 32, further characterized in that the ORFlab sequence of the SARS-CoV-2 virus has a length of 921 base pairs.

47. The rapid diagnostic kit for the detection of the SARS-CoV-2 virus according to claim 46, further characterized in that the ORFlab sequence of the SARS-CoV-2 virus corresponds to the sequence SEQ ID NO:

7.

48. A rapid diagnostic method for the detection of the SARS-CoV-2 virus, 5 characterized in that it comprises: a) taking a sample of the patient's exudate; b) denaturing the patient's exudate sample to obtain SARS-CoV-2 virus RNA; c) detecting the presence of the SARS-CoV-2 virus using ORFlab synthetic sequences specific for the SARS-CoV-2 virus; and d) determining whether the test result is negative or positive.

49. The rapid diagnostic method for the detection of the SARS-CoV-2 virus according to claim 48, further characterized in that the exudate is a pharyngeal exudate.

50. The rapid diagnostic method for the detection of the SARS-CoV-2 virus according to claim 48, further characterized in that the presence of the SARS-CoV-2 virus is detected by means of a pH-based colorimetric indicator. 15 51. The rapid diagnostic method for the detection of the SARS-CoV-2 virus according to claim 50, further characterized in that the colorimetric indicator is selected from hydroxynaphthol blue, phenol red, or a mixture thereof.