RU2769572C1 - Test system for detecting specific nucleotide sequences characteristic of the detected microorganisms or viruses, method for application of the test system (options) - Google Patents

Abstract

FIELD: molecular biology.

SUBSTANCE: invention relates to the field of molecular biology, virology and biotechnology. A test system is described for detecting specific nucleotide sequences characteristic of the detected microorganisms or viruses by amplification of nucleic acids of microbes or viruses, containing DNA polymerase with chain-displacing activity, primers for loop isothermal amplification, deoxyribonucleotides, salts, as well as detergent Triton™ X-100, bovine serum albumin, dithiothreitol, ethylenediaminetetraacetic acid, while the concentration of substances in the final liquid for the nucleic acid amplification reaction is: detergent Triton™ X-100, from 10 to 150 g/l, bovine serum albumin from 1 to 20 g/l, dithiothreitol from 6.25 to 18.75 mmol/l, ethylenediaminetetraacetic acid from 0.5 to 1.5 mmol/l. Also described is a test system for detecting specific nucleotide sequences characteristic of the detected microorganisms or viruses, by amplification of nucleic acids of microbes or viruses, by amplification of nucleic acids of microbes or viruses, containing DNA polymerase with chain-displacing activity, reverse transcriptase, primers for loop isothermal amplification, deoxyribonucleotides, salts, and detergent Triton™ X-100, bovine serum albumin, dithiothreitol, ethylenediaminetetraacetic acid, at the same time, the concentration of substances in the final liquid for the nucleic acid amplification reaction is: detergent Triton™ X-100 from 10 to 150 g/l, bovine serum albumin from 1 to 20 g/l, dithiothreitol from 6.25 to 18.75 mmol/l, ethylenediaminetetraacetic acid from 0.5 to 1.5 mmol/l. In addition, a method is presented for using these test systems to identify specific nucleotide sequences characteristic of the detected microorganisms or viruses by amplification of nucleic acids of microbes or viruses, where amplification of viral or microbial nucleic acids is performed by loop isothermal amplification at a temperature from 55 to 69°C.

EFFECT: invention can be used for amplification of nucleic acids (NC) of various origins.

9 cl, 16 dwg, 7 tbl, 15 ex

Description

Technical field

The invention relates to the field of molecular biology, virology and biotechnology. The invention can be used for amplification of nucleic acids (NA) of various origins.

Prior Art

The step of isolating, or extracting or purifying, nucleic acids is one of the longest and most labor-intensive steps in any nucleic acid analysis. In this regard, of great interest are methods and compositions that make it possible to reduce the duration and laboriousness of this process, in particular, methods for relatively rough and short lysis of a biological sample instead of a complete nucleic acid extraction process, and amplification methods that are compatible with such rough preparation of a biological sample. Among other things, of interest are methods for conducting loop isothermal amplification that are compatible with such preparation of a biological sample, since loop isothermal amplification has properties that are convenient for diagnosing pathogens: the speed of the reaction, the possibility of conducting it and evaluating the result without high-tech equipment, in particular, the evaluation of the result can be carried out visually, for example, by changing the color or cloudiness of the reaction mixture.

In addition, a common problem in molecular diagnostics and molecular biology in general is that the quality of the obtained samples decreases during storage and transportation. In particular, a significant problem is the risk of contact of the analyzed nucleic acids with nucleases present in the environment and the sample itself. However, if the goal is to analyze the nucleic acids of viruses (or other nucleoprotein particles), then due to the fact that viral NAs are securely packaged in the virion, the virus nucleic acids are most at risk of degradation during the isolation of NAs if the virion is already destroyed, and the nucleases are still not definitively inactivated.

Known patent RU2 252 964C2 "Method and kit for the synthesis of a nucleic acid having a nucleotide sequence, where complementary nucleotide sequences are alternately linked in one chain", describing the loop isothermal amplification reaction in general terms, without specifying the composition of the reaction mixture in such a way that this composition makes an optional step for isolating nucleic acids.

A number of analogues of the present invention are known, which allow amplifying nucleic acids from a poorly purified or unpurified sample.

Known patent EP 1 476 573 B1 "Rough biological derivatives suitable for the detection of nucleic acids", describing the conduct of NA amplification (namely, reverse transcription) using a coarse lysate of a biological sample as a source of matrix NA. In the proposed solution, a proteinase is used to obtain a lysate, which increases the cost of the reaction mixture, and also requires the proteinase to be inactivated by heating before amplification.

Also known is the patent EP 3642359 A1 "Methods for detecting target DNA directly in coarse samples by polymerase chain reaction and genotyping by high resolution melting curve analysis", describing the coarse lysis of a biological sample using proteinases, subsequent amplification of NA in polymerase chain reaction (PCR) and analysis of melting curves with high resolution. The technical solution described in this patent has the same disadvantages.

Known patent EP 3334533 A1 "Multiplex detection of target nucleic acids directly in samples containing blood", which involves adding blood directly to the amplification mixture, while excitation with radiation with a wavelength of more than 620 nm is used to detect nucleic acids in the sample, however, the patent describes not the composition of the amplification mixture, but overcoming the effect of blood, which makes it difficult to read fluorescence, in addition, amplification by PCR (rather than LAMP) is assumed, which uses high temperatures (and thermostable polymerases), which facilitates sample lysis, so it is relatively simple to choose the composition of the reaction mixture, providing both sample lysis and amplification, while LAMP requires a different composition of the reaction mixture.

Also known is US patent 2019/0078154 "Methods and reagents for polymerase chain reaction combined with reverse transcription" for a method for amplifying an RNA molecule contained in a biological sample by reverse transcription polymerase chain reaction (RT-PCR), in which RT-PCR carried out in a solution including: a polar aprotic solvent, serum albumin and optionally a non-ionic surfactant and/or a betaine variant of RT-PCR, in which the biological sample does not undergo the RNA purification step. As noted above, PCR and RT-PCR put forward different requirements for the reaction mixture, including the reaction mixture that is capable of lysing the biological sample placed in it than LAMP.

Known patent US 6,825,340 B2 "Methods and reagents for the inactivation of ribonucleases", describing the inactivation of RNase A, RNase 1 and/or RNase T1 in a cell extract using a combination of exposure to a reducing agent with heating. This patent focuses only on the inactivation of RNases, and not on the method of combining the lysis of a biological sample or the isolation of nucleic acids and the amplification of the resulting nucleic acids in one tube.

The closest analogue of the present invention is the international patent application WO 2020025947 A1 "Methods and compositions for the detection of microbes and viral particles" for a method of preparing a sample obtained from a subject, which may contain one or more microbes or viral particles, for direct amplification of a microbial or viral nucleic acid polymerase. The method consists in heating the vessel to a temperature of at least 70°C in the presence of a reagent that includes a detergent, a solvent, and one or more nucleic acid polymerases. This method requires heating the sample to a temperature of at least 70°C, which is not optimal for many amplification options, in particular, many isothermal amplification options, and requires the use of a thermostable polymerase.

In addition, high temperature increases the likelihood of destruction of NCs, especially RNA. For this reason, said patent application in its preferred embodiment describes heating the sample at the specified temperature for no more than 60 seconds, and in a particularly preferred embodiment, no more than 1.5 seconds. This approach is inconvenient, because requires changing the settings of the thermostat or other heater used, or using two heaters using different heating temperatures. In addition, the applicability of this approach to loop isothermal amplification (LAMP) is limited, since for LAMP, the most preferred and frequently used temperature is 60-65ºC rather than 70ºC.

Disclosure of invention

The objective of the present invention is to create a fast and reliable method capable of detecting the target analyte, namely viral or microbial NK, using one reaction mixture and in one tube.

The technical problem is solved due to the fact that a method for amplifying the nucleic acids of microorganisms or viruses with a DNA genome has been created, which consists in the fact that the sample is placed in a reaction mixture containing a DNA polymerase with a chain-displacing activity, primers for loop isothermal amplification, deoxyribonucleotides, salts, as well as Triton™ X-100 detergent, bovine serum albumin, dithiothreitol, ethylenediaminetetraacetic acid, and incubated at a temperature selected in the range from 50 to 69ºС in one test tube, after which the amplification result is detected, while the final mixture formed when mixing the sample with the reaction mixture, contains:

detergent Triton™ X-100 - from 10 to 150 g/l,

bovine serum albumin - from 1 to 20 g / l,

dithiothreitol - from 6.25 to 18.75 mmol / l,

ethylenediaminetetraacetic acid - from 0.5 to 1.5 mmol/l (option 1). To ensure a fluorescent determination of the amplification result, the concentration of bovine serum albumin is used in the optimal range for the reaction to proceed from 5 to 20 g/l, and the detection of the result of the amplification reaction is carried out by registering fluorescence, for which a fluorescent dye is additionally introduced into the reaction mixture, and the detection of the result of the amplification reaction nucleic acids of interest are carried out visually. To provide a visual determination of the result of amplification using a pH-sensitive dye, the pH of the reaction mixture is in the range from 8.0 to 10.0, and a concentration of 2-amino-2-hydroxymethyl-propane-1,3-diol is used to maintain the buffer capacity. more than 500 µmol/l, while the concentration of bovine serum albumin is in the range from 1 to 5 g/l, providing better detection of color change. In the embodiment for stable storage at room temperature, part of the components of the reaction mixture is dried, and part is in the activator solution, while the components of the reaction mixture are separated and the nucleic acid amplification reaction is started by combining the dried components, the activator solution and the sample.

According to the second variant, a method for amplifying nucleic acids of microorganisms or viruses with an RNA genome was created, which consists in placing the sample into a reaction mixture containing DNA polymerase with a chain-displacing activity, reverse transcriptase, primers for loop isothermal amplification, deoxyribonucleotides, salts, and also contains: Triton™ X-100 detergent, bovine serum albumin, dithiothreitol, ethylenediaminetetraacetic acid, and incubated at a temperature selected in the range from 50 to 69ºС in one test tube, after which the amplification (incubation) result is detected, and the final the mixture formed by mixing the sample with the reaction mixture contains:

detergent Triton™ X-100 - from 10 to 150 g/l,

bovine serum albumin - from 1 to 20 g / l,

dithiothreitol - from 6.25 to 18.75 mmol / l,

ethylenediaminetetraacetic acid - from 0.5 to 1.5 mmol/l (option 2), while the reaction mixture additionally contains an RNase inhibitor of non-protein nature, and the RNase inhibitor is polyvinyl sulfonic acid or its salt. To ensure fluorescent determination of the amplification result, the concentration of bovine serum albumin is in the optimal range for the reaction to proceed from 5 to 20 g/l, and the detection of the result of the amplification reaction of nucleic acids of interest is carried out by registering fluorescence, for which a fluorescent dye is additionally added to the reaction mixture , and detection of the result of the amplification reaction of the nucleic acids of interest is carried out visually. To ensure visual determination of the result of amplification using a pH-sensitive dye, the pH of the reaction mixture is used in the range from 8.0 to 10.0, and to maintain the buffer capacity, a concentration of 2-amino-2-hydroxymethyl-propane-1,3-diol is used, not more than 500 µmol/l, while the concentration of bovine serum albumin is in the range from 1 to 5 g/l, providing better detection of color change. In the embodiment for stable storage at room temperature, part of the components of the reaction mixture is dried, and part is in the activator solution, and the components of the reaction mixture are separated and the nucleic acid amplification reaction is started by combining the dried components, the activator solution and the sample.

Test systems for the detection of microorganisms or viruses have been created, which consist in using the created amplification method, and to ensure the reliability of the results obtained, the test systems are equipped with positive and negative control samples, as well as instructions for use, including rules for interpreting the results of the analysis.

Brief description of the figures

Fig. 1. Accumulation curves of the fluorescent amplification product and their dependence on the content of Triton™X-100 in the reaction mixture. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 8). Fluorescent product accumulation curves in the order they crossed the threshold line, from left to right: curves for the reaction mixture containing, respectively, 1) 100, 2) 50, 3) 20, 4) 10, 5) 5, 6) 1 and 7) 0 g/ l Triton™X-100.

Fig. 1a. Accumulation curves of the fluorescent amplification product and their dependence on the content of Triton™X-100 in the reaction mixture. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 4). Curves of accumulation of fluorescent product in the order they crossed the threshold line, from left to right: curves for the reaction mixture containing respectively 1) 100, 2) 125, 3) 150 g/l Triton™X-100.

Fig. 2. Accumulation curves of the fluorescent amplification product and their dependence on the content of BSA in the reaction mixture. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 7). Fluorescent product accumulation curves in the order they crossed the threshold line, from left to right: curves for the reaction mixture containing, respectively, 1) 20, 2) 15, 3) 10, 4) 5, 5) 2.5 and 6) 0 g/l BSA .

Fig. 3. Accumulation curves of the fluorescent amplification product and their dependence on the EDTA content in the reaction mixture. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 7). Fluorescent product accumulation curves in the order they crossed the threshold line, from left to right: curves for the reaction mixture containing, respectively, 1) 0.75, 2) 1, 3) 0.5, 4) 1.5, 5) 0.25 and 6 ) 2 mmol/L EDTA. Below the threshold line are the curves obtained for the negative control samples.

Fig. 4. Accumulation curves of the fluorescent amplification product and their dependence on the content of guanidine hydrochloride in the reaction mixture. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 7). Fluorescent product accumulation curves in the order they crossed the threshold line, from left to right: curves for the reaction mixture containing 1) 40, 2) 50, 3) 60, 4) 30, 5) 20, and 6) 0 mmol/L guanidine hydrochloride, respectively.

Fig. 5. Accumulation curves of the fluorescent amplification product and their dependence on the content of dithiothreitol in the reaction mixture. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 8). Fluorescent product accumulation curves in the order they crossed the threshold line, from left to right: curves for the reaction mixture containing, respectively, 1) 18.75, 2) 12.5, 3) 6.25, 4) 25, 5) 37.5, 6 ) 50 and 7) 0 mmol/l dithiothreitol.

Fig. 6. Accumulation curves of the fluorescent amplification product and their dependence on the content of polyvinyl sulfonic acid in the reaction mixture. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 8). Fluorescent product accumulation curves in the order they crossed the threshold line, from left to right: curves for the reaction mixture containing, respectively, 1) 0.00075; 2) 0.003; 3) 0.0003; 4) 0.0015; fifty; 6) 0.015 and 7) 0.06% polyvinyl sulfonic acid (mass fraction).

Fig. 7. Results of analysis of a series of clinical samples for the presence of the SARS-CoV-2 virus using the developed test system. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 3). S-shaped (sigmoid) curves (an example of such a curve is indicated by the number 1) crossing the threshold line and indicating exponential accumulation of the amplification product - specific amplification curves that occur in the presence of the SARS-CoV-2 virus or a specially designed positive control sample. Flat curves (an example of such a curve is indicated by the number 2) below the threshold line result from indolent non-specific amplification of nucleic acids in samples that do not contain the virus and are not counted as a positive test result.

Fig. 8. An example of fluorescent product accumulation curves that should be taken into account as a positive result when analyzing for the presence of the SARS-CoV-2 virus by the developed test system (an example of a reference curve is given, close in shape to an ideal sigmoid curve). The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 2). S-shaped (sigmoid) curve (indicated by the number 1) crossing the threshold line and indicating the exponential accumulation of the amplification product - a specific amplification curve that occurs in the presence of a pathogen or a specially designed positive control sample.

Fig. 9. Specificity of detection of the SARS-CoV-2 virus by the developed test system. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 3). The only curve that crosses the threshold line (indicated by the number 1) is the accumulation curve of the amplification product of the nucleotide sequence specific for the SARS-CoV-2 virus and included in the positive control sample. The number 2 indicates an example of a curve that occurs when analyzing a biological sample developed by the test system that contains a respiratory pathogen other than the SARS-CoV-2 virus.

Fig. 10. Specificity of detection of the SARS-CoV-2 virus by the developed test system with colorimetric detection of the amplification result. The reaction mixture changes color when incubated with a positive control sample whose nucleotide sequence matches the nucleotide sequence from the genome of the SARS-CoV-2 virus, but does not change color when incubated with samples corresponding to microorganisms and viruses other than SARS-CoV-2 (in black). -white image, this can be seen from the clearing of the mixture only in the test tube "K +").

Fig. 11. Comparison of the developed test systems with fluorescent and colorimetric detection of the amplification result with analogues manufactured by New England Biolabs. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 5). Fluorescent product accumulation curves in the order they crossed the threshold line, from left to right: 1) for the variant of the test system with fluorescent detection; 2) for the variant of the test system with colorimetric detection with additionally added fluorescent dye SYBR® Green I; 3) for an Isothermal Amplification Buffer Pack reaction mixture composed of Bst 2.0 WarmStart™ DNA Polymerase and 10X Isothermal Amplification Buffer (NewEnglandBiolabs); 4) for the reaction mixture WarmStart ^® Colorimetric LAMP 2X Master Mix (DNA & RNA) ("NewEnglandBiolabs") with additionally added fluorescent dye SYBR® Green I.

Fig. 12. Detection of E. coli bacteria using the reaction mixture included in the test system with fluorescent detection of the amplification result and specific primers. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 6). Fluorescent product accumulation curves in the order they crossed the threshold line, from left to right: accumulation of the amplification product for dilutions 1) 10 ⁶ , 2) 10 ⁵ , 3) 10 ⁴ CFU/ml; 4) the curve for 10 ³ cfu/ml is below the threshold line, as is 5) the curve for the negative control sample.

Fig. 13. Specific detection of influenza virus strain H1N1. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 4). The only curve that crosses the threshold line (indicated by the number 1) is the accumulation curve of the amplification product of the nucleotide sequence specific for the H1N1 influenza virus strain. The curves below the threshold line (2, 3) correspond to test tubes where other influenza viruses were introduced.

Fig. 14. Specific detection of influenza virus strain H3N2. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 5). The only curve that crosses the threshold line (indicated by the number 1) is the accumulation curve of the amplification product of the nucleotide sequence specific for the influenza virus strain H3N2. The curves below the threshold line correspond to test tubes where other influenza viruses (2, 3) and a negative control sample (4) were added.

Fig. 15. Specific detection of influenza B virus. On the horizontal axis - amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 3). The only curve that crosses the threshold line (indicated by the number 1) is the accumulation curve of the amplification product of the nucleotide sequence specific for influenza B virus. The curves below the threshold line correspond to test tubes where other influenza viruses were introduced.

Fig. 16. Evidence of the effectiveness of the test system with fluorescent detection of the amplification result after vacuum drying of the reagents included in its composition. The horizontal axis shows amplification cycles. The vertical axis shows relative units of fluorescence. The horizontal line parallel to the horizontal axis is the threshold line (indicated by the number 3). The only curve that crosses the threshold line (indicated by the number 1) is the accumulation curve of the product of amplification of the NK sequence specific for the SARS-CoV-2 virus, obtained from the analysis of a clinical sample known to be positive for SARS-CoV-2. The curves below the threshold line (2) correspond to the test tubes into which SARS-CoV-2 negative clinical specimens were added.

Implementation of the invention

A method has been developed for amplifying viral or microbial nucleic acids contained in a biological sample using a reaction mixture developed by the authors and having the necessary properties for the destruction of nucleoprotein particles, lysis of a biological sample and inactivation of nucleases simultaneously with the amplification of the target NA, a method for using such a reaction mixture and a test system based on this reaction mixture. A method has been developed for using the reaction mixture, including methods for detecting the amplification of the target NA: fluorescent and colorimetric, including visual. Test systems (variants) based on the developed reaction mixture have been developed, using both fluorescent and colorimetric methods for detecting the target NA, namely the NA of the pathogenic SARS-CoV-2 virus, the causative agent of the COVID-19 disease.

The proposed reaction mixture includes Triton™ X-100 detergent and dithiothreitol reducing agent, which are used in this reaction mixture, in addition to their standard use in amplification mixtures (protection of mixture components from aggregation for detergents, protection of mixture components from oxidation for reducing agents), for lysis of a biological sample and destruction of nucleoprotein, in particular viral, particles. Their content in the reaction mixture was selected in such a way that, in combination with heating to the amplification temperature, they caused partial or complete denaturation of the sample proteins and the release of NA from the sample. At the same time, their content in the reaction mixture was chosen so that, in combination with heating to the amplification temperature, it would not cause denaturation of Bst DNA polymerase and thus not disrupt the NA amplification process (which can be judged by the fact that the selected concentrations of these substances do not shift the threshold cycle increase, which would correspond to a shift to the right of the amplification curves on the graph, and vice versa, contribute to the optimization of amplification, which is expressed in a faster accumulation of the amplification product and a shift of the amplification curves to the left).

The selection of such optimal concentrations became possible because the Geobacillus stearothermophilus bacterium, from which the Bst polymerase is isolated, is thermophilic, as a result of which its proteins are more stable in the high temperature range (the temperature range of amplification using Bst polymerase) than the proteins of clinically significant biological samples, including proteins of human cells, as well as microbes and viruses affecting humans.

Also in the reaction mixture according to the invention, bovine serum albumin (BSA) is used to improve amplification parameters. The content of all these components, as well as EDTA (ethylenediaminetetraacetic acid) is optimized for the best amplification.

In addition, the reaction mixture contains the components necessary for loop isothermal amplification of NA: DNA polymerase with chain-displacing activity, primers, deoxyribonucleotides (deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and deoxythymidine triphosphate), salts.

It was also found that the addition of guanidine up to a concentration of 60 mmol/l improves the amplification of the target NA in the created reaction mixture, however, guanidine was not included in its composition, since the NA preparation often turns out to be contaminated with guanidine during the isolation of nucleic acids. , which introduces guanidine into the reaction mixture in the event that the user uses a preparation of isolated NAs as a sample, and not directly a biological sample.

A method is also proposed for using the developed reaction mixture, namely the amplification method, in which the developed reaction mixture is incubated at a constant temperature selected in the range from 50 to 69ºC, in any heating device capable of maintaining the selected incubation temperature with an accuracy of 0.5ºC, for the time best suited for the accumulation of predominantly specific amplification product in an amount compatible with the chosen method of detection of amplification products, i.e. usually not less than 15, but not more than 30 minutes.

Particular cases of the developed reaction mixture are variants of the reaction mixture designed for specific methods for detecting DNA amplification products. Thus, to detect DNA amplification products in the fluorescent version of their detection, the ability of the fluorescent dye SYBR® GreenI to bind to double-stranded DNA and produce a stronger fluorescence signal in this state than in the unbound form is used. The characteristics of this dye are the preferred wavelength of the radiation used for excitation, which is 497 nm, and the preferred wavelength of the detected radiation is 520 nm. Instead of SYBR® GreenI, another fluorescent DNA-binding dye known to those skilled in the art of molecular biology may be used.

To enable colorimetric pH-dependent detection of amplification results, the reaction mixture is prepared with a low content of tris(hydroxymethyl)aminomethane and, as a result, with a low buffer capacity. During the synthesis of the NC chain, protons are formed as a by-product, and due to the above features of the reaction mixture, this leads to a shift in the pH of the reaction liquid, which can be detected using pH-sensitive dyes, such as phenol red, as a result of which it is possible to determine the color change of the solution visually or with a colorimeter.

Other methods for detecting NA amplification known to those skilled in the art of molecular biology and specifically in the field of LAMP may also be used, for example, alkaline earth metal ion indicators such as hydroxynaphthol blue, or turbidimetric methods.

The advantage of the proposed invention is that it does not use the lysis of a biological sample using a proteinase (a typical solution to similar problems), which reduces the cost and simplifies the technical solution, in particular, eliminates the need for heating the reaction mixture at a high temperature before the amplification step to inactivate the proteinase. This makes the reaction mixture compatible with a heating source set to only one temperature and suitable for use in isothermal amplification techniques.

The proposed solution combines the destruction of a biological sample, the disassembly of virions or other nucleoprotein particles, the inactivation of nucleases and the amplification of NA in the same tube, at the same time interval and using the same mixture, thus acting simultaneously as a lysis buffer solution, a system for isolating nucleic acids, and an amplification reaction mixture. In this case, the polymerase competes with nucleases for nucleic acids, which makes it possible to more successfully amplify nucleic acids - with a lower risk that template NAs will be destroyed by nucleases even before amplification begins and will not be detected during analysis.

The proposed technical solution allows the amplification reaction, namely the loop isothermal amplification reaction (LAMP), using those samples that were taken even in such a simple transport medium as saline (0.9% sodium chloride). At the same time, the components of the reaction mixture (RS) make it possible to inactivate the nucleases contained in the sample directly during the amplification reaction.

In the variant for the analysis of the presence of viruses with an RNA genome, for better preservation of RNA in the reaction mixture, a non-protein thermostable inhibitor of ribonucleases (RNases), for example, polyvinyl sulfonic acid, is used. The reaction mixture for analysis of a sample for the content of RNA viruses also contains a reverse transcriptase for cDNA synthesis, which can then serve as a template for Bst DNA polymerase .

Particular cases of using the developed amplification method are the developed test systems for detecting RNA of the coronavirus SARS-CoV-2, the causative agent of the respiratory disease COVID-19. Coronavirus RNA is covered with several envelopes, lipid and protein, for this reason, the destruction of the capsid of coronaviruses and the release of genomic RNA from it under relatively mild temperature and chemical conditions (relatively mild conditions here mean conditions suitable for amplifying the released RNA by loop isothermal amplification) is rather complex technical problem, which is successfully solved by the developed test system.

In the case of dangerous viruses, such as SARS-CoV-2, the advantage of the developed reaction mixture is the ability to neutralize viruses during use. This ability of the mixture is evidenced by world experience using similar mixtures and similar temperature conditions, in particular, the chemical and temperature conditions used meet the conditions recommended by the US Centers for Disease Control and Prevention for samples obtained from patients with suspected viral hemorrhagic fever, for example, fever Ebola [Interim Guidance for the Management of Patients with Suspected Viral Hemorrhagic Fever in US Hospitals, http://www.cdc.gov/HAI/pdfs/bbp/VHFinterimGuidance05_19_05.pdf]. Moreover, a 30-minute incubation of a sample with Triton™X-100 detergent at room temperature, taken separately from heat, has also been demonstrated [Public Health England, "Testing methods for inactivating SARS-CoV-2: an interim report", June 12, 2020, https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/898605/HCM-CoV2-006-v3_Triton_X-100_TCF.pdf; also E.I. Patterson et al. “Methods for inactivating SARS-CoV-2 for further biological research”, bioRxiv, 2020] or heating at 65°C (the temperature used by the developed test system) for 10 minutes without detergent [DarnellME, TaylorDR. "Evaluation of methods for inactivation of severe acute respiratory syndrome coronavirus in non-cellular blood derivatives", Transfusion journal, 2006 Oct; 46(10): 1770–1777] to kill coronaviruses (SARS-CoV-2 and SARS-CoV) effectively enough that infectious viral particles cannot be detected in the sample by virological methods.

Test systems have been developed in which the developed method for amplifying viral or microbial nucleic acids contained in a biological sample using the proposed reaction mixture is provided with a positive and negative control sample so that the user can verify that a specific amplification reaction occurs in this setting. In addition, in these test systems, the enzymes or primers necessary for the loop isothermal amplification reaction are separated from the main reaction mixture in order to avoid nonspecific reactions before the sample is added, as well as to increase the stability of the components during storage. As an alternative, versions of test systems have been developed in which most of the components are subjected to vacuum drying or lyophilization, and the remaining components are included in the diluent buffer solution.

The invention is confirmed by examples.

The selection of the optimal concentrations of the components of the reaction mixture, universal for the amplification of NC microorganisms and viruses with both DNA and RNA genome, is described in examples 1-5. Example 6 describes the study of the effect of polyvinyl sulfonic acid, an RNase inhibitor, which can be used to analyze the content of RNA genomes of interest for better preservation.

Example 1

Reaction mixture optimization for Triton™ X-100 component

In the reaction mixture for LAMP, the content of the Triton™ X-100 component was varied and a reaction was performed for the detection of SARS-CoV-2 coronavirus RNA. The resulting curves (Fig. 1, 1a) were compared by threshold cycles, the concentration of the component at which the threshold cycle was the smallest was taken as optimal.

The results were compared for concentration ranges from 0 to 100 g/L Triton™ X-100 (in the final reaction mixture resulting from the addition of the sample) and also, in a separate set of experiments, from 100 to 150 g/L. The best results in both cases were obtained using a concentration of 100 g/l, concentrations in the range from 10 to 100 g/l and from 100 to 150 g/l also led to a fairly rapid accumulation of the specific amplification product, which is displayed as a rise in the curve.

Example 2

Optimization of the reaction mixture for the component of bovine serum albumin (BSA)

In the reaction mixture for LAMP, the content of the BSA component was varied and a reaction was performed for the detection of SARS-CoV-2 coronavirus. The resulting curves (Fig. 2) were compared by threshold cycles, the concentration of the component at which the threshold cycle was the smallest was taken as optimal.

BSA improved threshold cycle score at all concentrations tested from 1 to 20 g/l compared to the formula without BSA. The best results were obtained using a concentration of 20 g/l (in the final reaction mixture obtained after the addition of the sample), however, the downward shift in the threshold cycle was rather small compared to concentrations of 15 and 10 g/l, while at high concentrations BSA changed the color of the reaction mixture, which reduces its suitability for visual colorimetric determination of the amplification result, as well as the stability of the mixture during freezing-thawing (precipitation precipitated), so it was decided not to further increase the concentration of BSA. Thus, the BSA concentration range from 5 to 20 g/l was recognized as the most favorable for amplification, and from 1 to 5 g/l for visual colorimetric determination of the amplification result.

Example 3

Optimization of the reaction mixture for the EDTA component

In the reaction mixture for LAMP, the content of the EDTA component was varied and a reaction was performed for the detection of SARS-CoV-2 coronavirus. The resulting curves (Fig. 3) were compared by threshold cycles, the concentration of the component at which the threshold cycle was the smallest was taken as optimal.

The best results were obtained using a concentration of 0.75 mmol/l (in the final reaction mixture obtained after the addition of the sample), the closest studied values (from 0.5 to 1.5 mmol/l) were suboptimal.

Example 4

Compatibility of the reaction mixture with the presence of guanidine hydrochloride in it

In the reaction mixture for LAMP, the content of guanidine hydrochloride was varied and a reaction was carried out for the detection of SARS-CoV-2 coronavirus. The resulting curves (Fig. 4) were compared by threshold cycles, the concentration of the component at which the threshold cycle was the smallest was taken as optimal.

The best results were obtained using a concentration of 40 mmol/l (in the final reaction mixture obtained after the addition of the sample), the closest studied values were suboptimal.

Thus, it was found that the addition of guanidine up to a concentration of 60 mmol/l improves the amplification of the target NA in the created reaction mixture; , if the user uses a preparation of isolated NAs as a sample, and not directly a biological sample, therefore, guanidine was not used in the developed reaction mixture in the future.

Example 5

Optimization of the reaction mixture for the dithiothreitol component

In the reaction mixture for LAMP, the content of dithiothreitol was varied and a reaction was performed for the detection of SARS-CoV-2 coronavirus. The resulting curves (Fig. 5) were compared by threshold cycles, the concentration of the component at which the threshold cycle was the smallest was taken as optimal.

The best results were obtained using a concentration of 18.75 mmol/l of dithiothreitol (in the final reaction mixture obtained after the addition of the sample), the closest studied values (from 6.25 to 12.5 mmol/l) were suboptimal.

Example 6

Study of the possibility of using polyvinyl sulfonic acid for the inhibition of ribonucleases

In the reaction mixture for LAMP, the content of polyvinyl sulfonic acid was varied and a reaction was performed for the detection of SARS-CoV-2 coronavirus. The resulting curves (Fig. 6) were compared by threshold cycles, the concentration of the component at which the threshold cycle was the smallest was taken as optimal.

At the maximum concentration used, inhibition of the amplification reaction was observed. In reaction mixtures with all other tested concentrations, amplification proceeded at approximately the same rate, and no inhibition of amplification was observed, which allows using this RNase inhibitor as an additional (in relation to the effect of heating in combination with Triton™ X-100 detergent and dithiothreitol reducing agent) protection RNAs of interest from RNases.

Thus, by optimizing the content of the individual components of the reaction mixture, as described in examples 1-5, and choosing the concentration of each of these components that turned out to be optimal in each of the described experiments, we obtained a reaction mixture suitable for the rapid and reliable detection of pathogens directly in biological samples (optimized concentrations of the components are shown in table 1).

Table 1. Composition of the optimized reaction mixture

Component Concentration detergent Triton™ X-100 from 10 to 150 g/l bovine serum albumin from 1 to 20 g/l dithiothreitol from 6.25 to 18.75 mmol/l ethylenediaminetetraacetic acid 0.5 to 1.5 mmol/l

By adding polyvinyl sulfonic acid to this mixture at the concentration found optimal in Example 6, a reaction mixture can be obtained that is even more suitable for analyzing a sample for pathogens with an RNA genome. In addition, to analyze a sample for pathogens with an RNA genome, it is necessary to add reverse transcriptase to the reaction mixture, which was taken into account in the further development of test systems.

The operating temperature range of Bst polymerase allows its use at the incubation temperature of the reaction mixture from 50 to 72ºС, however, the preferred temperature range is 60-69ºС, which makes it possible to optimally combine the stability of the enzyme and the speed of its operation (and, therefore, reduce the analysis). Within this range, an amplification temperature of 65°C was chosen for further use.

Further examples describe the use of the proposed amplification method as part of test systems for the detection of specific microorganisms or viruses. Test systems include positive and negative control samples and user instructions that describe the rules for setting up the analysis and interpreting the results of the study. Successful and specific detection in the sample of both DNA (Example 13) and RNA genomes (Examples 8-12, 14-15) of interest is shown.

Example 7

Preparation of samples for analysis for the content of the respiratory virus, which is the target analyte, using the proposed amplification method

To prepare the sample for analysis, a swab from the nasopharynx and/or oropharynx was introduced into a test tube with 500 μl of physiological solution (0.9% sodium chloride solution), vigorous rotational movements ensured the separation of biological material from the probe/tampon into the solution, trying to prevent splashing potentially contaminated fluid, after which the probe / swab was deactivated, considering it as infectious. In the case of taking a nasopharyngeal and oropharyngeal swab from the same patient, the biological material from both probes / swabs was washed off in the same tube to increase the viral load. The samples were stored in a refrigerator at a temperature of 2 to 8°C and transported in compliance with the "cold chain". The transport medium and/or the sample storage medium was the saline solution in which the wash was performed.

This sample preparation provides an optimal concentration of virus in the sample, a low concentration of potential interfering substances, and does not change the pH of the sample. Keeping the pH of the sample in the physiological range is important for the variant of the reaction mixture with a pH-dependent dye, which provides colorimetric detection of the amplification result.

Example 8

Analysis of the sample for the content of the SARS-CoV-2 virus (pathogenic virus with an RNA genome) using the claimed reaction mixture as part of the test system in the version with colorimetric detection of the amplification result

To carry out the amplification reaction, a test system was used in the embodiment with colorimetric detection of the amplification result, which includes a reaction mixture ("RS") containing all the components necessary for amplification of viral RNA, except for primers and matrix NK, and optimized according to examples 1- 6, and "Primers" - a mixture of primers, as well as control samples: positive and negative control samples "K+" and "K-", which are, respectively, a solution of the PCR amplification product of a specific region of the SARS-CoV-2 genome and water.

For analysis, a solid-state thermostat was used, which allows heating the samples to 65°C, and microtubes with a volume of 0.5 to 1.5 ml, suitable for the thermostat sockets.

The components of the test system were thawed and thoroughly mixed. While working with the reagents, they were cooled by placing them on ice, a cold cell (cold accumulator) or in a chilled rack (for example, an isothermal rack Isofreeze®).

Microtubes were prepared in the amount of n+3, where n is the number of samples to be examined (three tubes are used as controls). They were placed to cool in an isothermal rack, pre-chilled at -20°C (preferably), or in ice. Another test tube was placed in an isothermal stand or ice to mix "RS" and "Primers".

A mixture of “RS” and “Primers” was prepared in the cold (in an isothermal rack or in ice) on the basis that 15 μl of “RS” and 2 μl of “Primers” are consumed for the analysis of one sample, with a margin of 10%, necessary to level out errors pipettes (see table 2). Stir the mixture with a pipette tip placed on the pipette. The resulting mixture was used immediately.

Table 2. Preparation of a mixture of "RS" and "Primers" for 20 samples, including controls. If it is necessary to analyze, for example, 30 samples, all volumes from the table are multiplied by 1.5, etc.

Component Volume "RS" 330 µl "Primers" 44 µl

17 μl of the mixture of “RS” and “Primers” were added to each microtube. Analyzed samples were added, “K-” and “K+” according to Table 3. After adding these components, the mixture was stirred in each test tube with a pipette tip without removing it from the pipette.

Table 3. Scheme of setting up the reaction in test and control microtubes

Component Test tube (take the required number of tubes and carry out the reaction in each) Colorimetric standard Negative
the control Positive control "RS"* 15 µl 15 µl 15 µl 15 µl "Primers"* 2 µl - 2 µl 2 µl Sample 3 µl - - - "TO-" - 3 µl 3 µl - "K+" - - - 3 µl Total volume 20 µl 18 µl 20 µl 20 µl Color before reaction Pink Pink Pink Pink What should be the color after the reaction If the sample has NA of the virus of interest: yellow.
If there is no NA of the virus of interest in the sample: pink. Pink Pink Yellow

*Can be prepared cold and immediately use the mixture of "PC" and "Primers" for all tubes at once, as described above in the text.

The microtubes were placed in a solid state thermostat preheated to 65°C.

After 25 min, all microtubes were taken out of the thermostat, including the colorimetric standard, allowed to cool to room temperature on the table (for 2–3 min), and the color of the liquid was visually assessed against a white background, compared with the colorimetric standard.

It is extremely important to incubate the microtubes in the incubator for no longer than the indicated time! Over time, non-specific amplification products may accumulate in the tubes, which can lead to a false positive result.

The liquid in the “colorimetric standard” tube remains pink, and the color of the liquid in the remaining tubes is compared with it.

A changed, yellow, color of the liquid means a positive test result (presence of NK virus) for the test sample. The pink color of the liquid means a negative test result (no NK virus) for the test sample. The results of the analysis of a batch of samples are valid only if the liquid in the tube where “K+” was added instead of saline with the sample turned yellow, and the liquid in the tube where “K-” was added instead of saline with the sample, stayed pink.

If the liquid in the tube where “K+” was added instead of saline with the sample remains pink, this means that the loop isothermal amplification reaction in this batch of samples did not pass, which threatens to give false negative results, so the results of the analysis of this batch of samples are considered unreliable .

If the liquid in the test tube, where “K-” was added instead of saline with the sample, turned yellow, this means contamination of reagents, consumables or the workplace with nucleic acids with a nucleotide sequence characteristic of the virus being detected, which threatens to give false positive results, therefore the results analyzes of this batch of samples are considered unreliable. It is required to take measures to identify and eliminate the source of contamination and repeat the analysis of all samples of this production, for which a positive result was obtained. Samples of this production, the analysis of which gave a negative result, can be considered as negative.

Example 9

Analysis of the sample for the content of the virus, which is the target analyte, using the claimed reaction mixture as part of the test system in the version with fluorescent detection of the amplification result

To carry out the amplification reaction, a test system with fluorescent detection of the amplification result was used, which includes a reaction mixture ("RS") containing all the components necessary for amplification, except for enzymes and matrix NK, and optimized according to examples 1-6, and "Enzyme "- a mixture of enzymes necessary for the amplification of viral RNA (reverse transcriptase and DNA polymerase Bst ), as well as control samples: positive and negative control samples "K +" and "K-", which are, respectively, a solution of the PCR product of amplification of a specific site SARS-CoV-2 genome and water.

For the analysis, we used amplifiers that allow heating samples to 65°С and recording fluorescence via the _SYBR (or FAM) _channel ; with the devices used.

The following amplification conditions were used: 25 minutes incubation, constant temperature 65°C.

During work with the reagents, they were cooled by placing them in a chilled rack (for example, an isothermal rack Isofreeze®), on a cold element (cold accumulator) or in ice.

The components of the Reagent Kit were thawed and thoroughly mixed (by pipetting or with 1-2 strokes on the tube wall).

Microtubes were prepared in the amount of n+2, where n is the number of samples to be examined, and 2 tubes are used as controls.

Microtubes were placed in an isothermal rack cooled at -20°C (preferred), or, in the absence of such, in ice. If the cooling mode is violated during operation, non-specific amplification products are most likely formed.

A mixture of "RS" and "Enzyme" was prepared in a separate chilled test tube according to Table 4, thoroughly mixed (by pipetting or with 1-2 strokes on the tube wall). The mixture was used immediately.

An automatic dispenser set at 8.5 μl was prepared with a tip on, the tip was cooled by dipping it for 5-10 seconds in a cold (standing in an isothermal rack or in ice) mixture of "RS" and "Enzyme".

Table 4. Preparation of a mixture of "RS" and "Enzyme" according to the number of analyzed samples *

Component Volume per 1 sample, µl Volume for 10 samples, µl Volume for 24 samples, µl Volume for 48 samples, µl Volume for 96 samples, µl "RS" 24 264 634 1267 2534 "Enzyme" 1.5 16.5 40 79 158

*To study any other number of samples (20, 30, 40…), the number of “RS” and “Enzyme” for 10 samples must be multiplied by the appropriate coefficient (2, 3, 4…).

The mixture of “RS” and “Enzyme” was divided between chilled microtubes for PCR so that 8.5 μl of the mixture was added to each microtube for PCR.

Reagents were added to microtubes according to Table 5.

Stir the mixture in each tube by tapping the tube with your finger.

Table 5. Scheme of setting up the reaction in test and control microtubes

Component Test microtube Negative
the control Positive control Mix "RS" + "Enzyme" 8.5 µl 8.5 µl 8.5 µl Sample 1.5 µl - - "TO-" - 1.5 µl - "K+" - - 1.5 µl Total volume 10 µl 10 µl 10 µl

The microtubes were immediately placed in the cycler and the amplification program was started.

In this case, for analysis, the cycler was prepared in advance in accordance with the cycler operation manual. The cycler was programmed according to the following principles:

• incubation temperature is constant and is 65°С;

• total incubation time of samples in the instrument should be 25 minutes. This time includes not only the cycle time in the cycler program, but also the time between cycles required to measure fluorescence. Those. 75 cycles of 20 seconds cannot be programmed, as the total incubation time will exceed 25 minutes, which may lead to non-specific amplification. The time required to transition between cycles, and thus the number of cycles required, will depend on the cycler model;

• Therefore, when the analysis was first started, it was measured how many cycles of 20 seconds (hereinafter referred to as N) would correspond to a total sample incubation time of 25 minutes, using a clock or a timer. For the CFX96 cycler, in the case of cycles of 20 seconds, this number of cycles N equals 40 cycles. In this case, it is necessary to stop the cycler program execution after 25 minutes;

• at further starts of the analysis, the required number of cycles is programmed, N cycles of 20 seconds, which was determined at the first start (see paragraph above), with fluorescence measurement on the SYBR channel (FAM) every 20 seconds.

Usually, after the amplification, a set of curves of various shapes is observed, as in Fig. 7, to which the following result interpretation criteria should apply:

For the negative control "K-": the value of the threshold cycle Ct in the SYBR (FAM) channel should not be determined (on a number of cyclers this is indicated as the threshold cycle Ct value of 0.00) or must be at least N cycles (in the case of cycles of 20 seconds ). If for "K-" the value of Ct is less than N cycles, this indicates the presence of contamination in the system. In this case, the results of this formulation are considered unreliable. It is required to take measures to identify and eliminate the source of contamination and repeat the analysis of all samples of this production, for which a positive result was obtained. Samples of this production, the analysis of which gave a negative result, should be counted as negative.

For positive control "K+": the Ct value in the SYBR channel (FAM) must be less than N cycles, otherwise the whole setting is considered invalid.

For each microtube: the Ct value in the SYBR (FAM) channel must be more than 6 cycles (20 seconds each). If in the first 2 minutes of the reaction (in the first 6 cycles of 20 seconds) amplification is detected (in other words, the fluorescence kinetic curve crosses the threshold line), such amplification is considered non-specific and is not taken into account in the analysis.

In addition, they are not taken into account, since they are non-specific, flat curves that do not resemble the typical curve obtained in PCR in shape (ie, a curve with an exponential accumulation of the amplification product, as in Fig. 8). In FIG. 7 they are under the threshold line, but even if they cross the threshold line, they are also not taken into account. Only characteristic kinetic growth curves of the S-shaped (sigmoid) fluorescence signal are taken into account, as in Fig. eight.

If the Ct value in the SYBR (FAM) channel is not detectable (on some cyclers this is referred to as a cycle threshold Ct value of 0.00) or is at least N cycles, the analyzed sample is considered negative, that is, it does not contain NA of the virus of interest.

If the Ct value in the SYBR channel (FAM) is less than N cycles, the analyzed sample is considered positive, that is, it contains the NA of the virus of interest.

Example 10

Evidence of the specific detection of SARS-CoV-2 virus RNA by a test system with fluorescent detection of the amplification result created on the basis of the proposed reaction mixture

To determine the analytical specificity of the test system, an analysis was performed, as in example 9, with samples of pathogens of respiratory infections other than SARS-CoV-2. The results of the analysis are shown in table 6.

A single fluorescent amplification product accumulation curve was observed corresponding to the positive control sample included in the test system (Fig. 9).

Table 6. Specific detection of SARS-CoV-2 virus RNA by a test system with fluorescent detection of the amplification result

Pathogen Sample No. threshold cycle number Result Streptococcus pneumoniae one - Not found Streptococcus pneumoniae one - Streptococcus pneumoniae one - Human metapneumovirus (virulent strain "HM-1") 2 - Not found Human metapneumovirus (virulent strain "HM-1") 2 - Human metapneumovirus (virulent strain "HM-1") 2 - Coronavirus (strain R/Leningrad/2/77) 3 - Not found Coronavirus (strain R/Leningrad/2/77) 3 - Coronavirus (strain R/Leningrad/2/77) 3 - Parainfluenza virus type 1 (strain "Sendai Biomed") 4 - Not found Parainfluenza virus type 1 (strain "Sendai Biomed") 4 - Parainfluenza virus type 1 (strain "Sendai Biomed") 4 - Human adenovirus A-5 (strain Adeno-5) 5 - Not found Human adenovirus A-5 (strain Adeno-5) 5 - Human adenovirus A-5 (strain Adeno-5) 5 - haemophilus influenzae 6 - Not found haemophilus influenzae 6 - haemophilus influenzae 6 - Infuenza A virus (H7N9)) 7 - Not found Infuenza A virus (H7N9)) 7 - Infuenza A virus (H7N9)) 7 - Infuenza A virus (H1N1)) eight - Not found Infuenza A virus (H1N1)) eight - Infuenza A virus (H1N1)) eight - Infuenza A virus (H3N2)) nine - Not found Infuenza A virus (H3N2)) nine - Infuenza A virus (H3N2)) nine - Influenza B virus ten - Not found Influenza B virus ten - Influenza B virus ten - Positive control sample 21.49 Revealed Negative control sample - Not found

Nasopharyngeal and oropharyngeal swabs from individuals without evidence of respiratory disease were used to determine diagnostic specificity. No nonspecific reactions were identified.

Example 11

Evidence of specific detection of SARS-CoV-2 virus RNA by a colorimetric detection test system based on the proposed reaction mixture

To determine the analytical specificity of the test system, an analysis was performed, as in example 8, with samples of pathogens of respiratory infections other than SARS-CoV-2. The results of the analysis are shown in table 7.

At the end of the reaction, a change in the color of the reaction mixture was observed only in the test tube, where a positive control sample, which was part of the test system, was introduced (Fig. 10).

Table 7. Specific detection of SARS-CoV-2 virus RNA by the developed test system with colorimetric detection of the fluorescence result

Pathogen Sample No. Color change Result Streptococcus pneumoniae one - Not found Human metapneumovirus (virulent strain "HM-1") 2 - Not found Coronavirus (strain R/Leningrad/2/77) 3 - Not found Parainfluenza virus type 1 (strain "Sendai Biomed") 4 - Not found Human adenovirus A-5 (strain Adeno-5) 5 - Not found haemophilus influenzae 6 - Not found Infuenza A virus (H7N9)) 7 - Not found Infuenza A virus (H1N1)) eight - Not found Infuenza A virus (H3N2)) nine - Not found Influenza B virus ten - Not found Positive control sample (K+) + Revealed Negative Control (K-) - Not found

Example 12

Comparison of the developed test systems with existing analogues

The specific amplification of coronavirus RNA for the developed test systems and similar products available on the market were compared: WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) and Isothermal Amplification Buffer Pack (NewEnglandBiolabs). The reaction mixture developed for the test system with colorimetric detection of the amplification result, as well as the WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) reaction mixture, was supplemented with SYBR® GreenI fluorescent dye for comparison with analogs using the principle of fluorescence detection: the following amount was added 50X (50x) SYBR® GreenI to achieve a 1X concentration in the reaction mixture after sample addition. The reaction was carried out as described in example 9, using as a template total RNA from patients with COVID-19, isolated from a swab from the oropharynx by the method of specific sorption on magnetic particles. Accumulation curves of fluorescent products were observed (Fig. 11), while the best (lowest) value of the threshold cycle was observed for the developed test system with fluorescent detection of the amplification result, in second place in this indicator was the developed test system designed for colorimetric detection of the amplification result .

Thus, the developed test systems are superior to those produced by NewEnglandBiolabs even in a situation where isolated coronavirus RNA serves as a reaction matrix (although the developed test systems were optimized primarily in the direction of the possibility of analyzing a biological sample without the RNA isolation step).

Example 13

Detection of E. coli bacteria (opportunistic pathogen with a DNA genome) by the proposed amplification method

A suspension of E. coli strain DH5α bacteria was subjected to serial 10-fold dilutions up to a content of 10 ³ CFU/ml and 1.5 μl of each dilution was added as a sample to the reaction mixture prepared as for a test system with fluorescent detection for the detection of the SARS virus -CoV-2, but with primers matched to the E. coli genome. Amplification product accumulation was observed (FIG. 12) for dilutions of 10 ⁶ , 10 ⁵ , 10 ⁴ cfu/ml but not for 10 ³ cfu/ml.

Example 14

Specific detection of influenza viruses (pathogenic viruses with RNA genome) by the proposed amplification method

Primers for LAMP were selected suitable for detection of influenza A virus H1N1, influenza A virus H3N2 or influenza B, respectively. but in each case with primers to detect one of these viruses. Observed specific amplification of viral NK in the presence of suitable primers and the absence of amplification when the primers and the strain of the virus did not match (Fig. 13-15).

Example 15

Vacuum drying of test system components

A mixture of components was prepared, which included:

1) a reaction mixture developed for the test system, modified so that a complete reaction mixture is formed only after combining with an activator buffer solution, and also

2) a mixture of enzymes "Enzyme" (a mixture of Bst DNA polymerase and reverse transcriptase from the same test system. This mixture of components was subjected to vacuum drying in a centrifugal evaporator in the presence of a lyoprotector (trehalose), stored for 2 months at room temperature, after then the dried components of the mixture were dissolved in the buffer solution-activator and the analysis of clinical samples was carried out with the obtained reagent.The positive control sample was dried without the addition of trehalose, for use it was dissolved in water for molecular biology.No false positive and false negative results of the analysis were observed (Fig. 16), which proves the applicability of the dried format of the developed diagnostic test system.

All the above examples prove the fulfillment of the technical task, namely, the creation of a fast and reliable method capable of detecting the target analyte, namely viral or microbial NK, using one reaction mixture and in one tube.

Industrial Applicability

All of the above examples confirm the industrial applicability of this invention.

List of abbreviations

NA - nucleic acid,

DNA is deoxyribonucleic acid,

PCR - polymerase chain reaction,

LAMP - loop isothermal amplification,

RNA is ribonucleic acid,

RT-PCR - reverse transcription combined with polymerase chain reaction,

RNase, ribonuclease

g - gram,

l - liter,

SARS-CoV-2, severe acute respiratory syndrome-associated coronavirus 2,

COVID-19 - coronavirus disease 2019,

BSA - bovine serum albumin,

EDTA - ethylenediaminetetraacetic acid,

SARS-CoV is a severe acute respiratory syndrome-associated coronavirus

CFU - colony forming units.

Claims (17)

1. Test system for the detection of specific nucleotide sequences characteristic of detected microorganisms or viruses by amplifying the nucleic acids of microbes or viruses, containing DNA polymerase with chain-displacing activity, primers for loop isothermal amplification, deoxyribonucleotides, salts, and Triton detergent ™ X-100, bovine serum albumin, dithiothreitol, ethylenediaminetetraacetic acid, while the concentration of substances in the final liquid for the nucleic acid amplification reaction is: detergent Triton™ X-100 - from 10 to 150 g/l, bovine serum albumin - from 1 to 20 g / l, dithiothreitol - from 6.25 to 18.75 mmol / l, ethylenediaminetetraacetic acid - from 0.5 to 1.5 mmol / l. 2. Test system for detection of specific nucleotide sequences characteristic of detected microorganisms or viruses by amplification of nucleic acids of microbes or viruses, containing DNA polymerase with chain-displacing activity, reverse transcriptase, primers for loop isothermal amplification, deoxyribonucleotides, salts, and also Triton™ X-100 detergent, bovine serum albumin, dithiothreitol, ethylenediaminetetraacetic acid, while the concentration of substances in the final liquid for the nucleic acid amplification reaction is: detergent Triton™ X-100 - from 10 to 150 g/l, bovine serum albumin - from 1 to 20 g / l, dithiothreitol - from 6.25 to 18.75 mmol / l, ethylenediaminetetraacetic acid - from 0.5 to 1.5 mmol / l. 3. The test system according to claim 1 or 2, which additionally contains an RNase inhibitor of a non-protein nature. 4. The test system according to claim 3, in which the RNase inhibitor is polyvinyl sulfonic acid or its salt. 5. Test system according to claim 1 or 2, which additionally contains a dye. 6. The test system according to claim 5, wherein the dye is a fluorescent dye. 7. The method of using the test system according to paragraphs. 1-6 to identify specific nucleotide sequences characteristic of the detected microorganisms or viruses by amplifying the nucleic acids of microbes or viruses, where the amplification of viral or microbial nucleic acids is carried out by the loop isothermal amplification method at a temperature of 55 to 69ºС. 8. The method of using the test system according to claim 7, in which the SARS-CoV-2 coronavirus is detected. 9. The method of using the test system according to claim 7, in which the influenza virus is detected.

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