RU2795460C1 - Method for determining dnase activity in real time - Google Patents

Abstract

FIELD: biotechnology; molecular biology.

SUBSTANCE: method for determining DNase activity is proposed, including the use of a substrate obtained by annealing two complementary oligonucleotides in the presence of the EVA Green intercalating dye, PCR in a mixture containing a DNA oligonucleotide substrate incubated with the EVA Green intercalating dye, an RNA-free sample, and a reaction buffer for DNase activity, including cofactors and a buffer system to maintain pH in the reaction mixture, the presence of non-specific DNase activity is determined by the real-time DNA hydrolysis reaction of the oligonucleotide substrate based on the fluorescence drop in the test sample relative to the control sample.

EFFECT: invention provides an extension of the arsenal of methods for determining the presence of DNase activity in samples for bacteriological diagnostics of microorganisms.

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Description

Technical field

The invention relates to biotechnology, in particular to molecular biology and medical microbiology, and can be used both in research and practical work to determine the presence of DNase activity in samples and bacteriological diagnosis of a number of microorganisms.

State of the art

DNases are enzymes that cleave DNA at phosphodiester bonds into oligonucleotide fragments or single nucleotides.

The test for the determination of DNase activity is common for the identification of pathogenic microorganisms [Gerceker et al., 2009].

A number of clinically significant eukaryotic and prokaryotic microorganisms produce DNases as virulence factors. These include gram-positive bacterial pathogens such as Staphylococcus aureus and streptococcal species such as Streptococcus pyogenes [Berends et al., 2010].

Currently, there are various methods and media for the determination of deoxyribinuclease (DNase) activity.

1. Methods Kalinichenko N.F., Ovcharenko O.I., Biryukova S.V. "On the determination of DNase in microorganisms" (ZHMEI, 1968, No. 12, pp. 11-14); Invention SU 1693058 A1 (Chisinau State Medical University) 11/23/1991 "Method for determining the DNase activity of microorganisms"; Invention RU 2684721 C1 (Federal State Budgetary Educational Institution of Higher Education "Pacific State Medical University" of the Ministry of Health of the Russian Federation (RU) 04/11/2019 "Nutritious medium for determining DNase activity in pathogenic gram-positive bacteria"; Order of the USSR Ministry of Health dated 04/22/1985 No. 535 "About the unification of microbiological (bacteriological) research methods used in clinical diagnostic laboratories of medical institutions, a medium for determining the DNase activity of bacteria is described"; Express method on agar plates proposed by [JEFFRIES, HOLTMAN, GUSE, 1957]; Schreier modified DNase test agar, which uses toluidine blue to detect DNase activity [Schreier, 1969]; Detection of nuclease activity in semisolid and broth cultures - PubMed,] to determine nuclease activity, and many other commercially available media containing DNA agar. These tests have a number of disadvantages, including a long time to review the results of the DNase activity of bacteria and the high cost of the components used.

2. Another analogue of the claimed invention in terms of the combination of features is the method presented by [Gerceker et al., 2009] as a rapid test for the detection of DNase activity of microorganisms: "DNase tube". In this method, bacteria are added to a broth medium containing DNA and DNA degradation caused by a DNase bacterium is monitored by running agarose gel electrophoresis. However, this method makes it possible to observe DNase activity in real time.

3. The closest analogue of the claimed invention in terms of the set of features is the method presented [E et al., 2019] in a study using fluorescently labeled oligonucleotides for the diagnosis of microorganisms producing nucleases, however, the researchers proposed self-locking hairpins, the use of which may not be commercially profitable.

The invention solves the problem of determining the presence of non-specific DNase activity in real time.

The task is solved due to the fact that:

1. It is proposed to analyze DNase activity on a substrate obtained by annealing two mutually complementary oligonucleotides (one oligonucleotide contains a fluorescent dye at the 5'-end, while a fluorescence quencher is located at the 3'-end of the second).

2. It is proposed to use supercoiled plasmid DNA as a substrate in the presence of an intercalating dye.

3. It is proposed to use oligonucleotides as a DNA substrate in the presence of an intercalating dye

The technical result of this invention is to determine the presence of non-specific DNase activity in real time.

To solve the technical problem and achieve the claimed technical result, it is proposed to analyze the DNase activity on a substrate obtained by annealing two complementary oligonucleotides (one oligonucleotide contains a fluorescent dye at the 5'-end, while a fluorescence quencher is located at the 3'-end of the second).

It is also proposed to use supercoiled plasmid DNA as a substrate in the presence of an intercalating dye.

It is also proposed to use oligonucleotides as a DNA substrate in the presence of an intercalating dye.

The technical result is achieved by using a substrate obtained by annealing two complementary oligonucleotides: one oligonucleotide contains a fluorescent dye at the 3'-end, while a fluorescence quencher is located at the 3'-end of the second. An equilibrium mixture of a pair of oligonucleotides in buffer containing (10 mM Tris-HCl pH 8.0, 25 mM NaCl) is heated in a boiling water bath for 3 minutes. A substrate of fluorescently labeled oligonucleotides, a sample, as well as a reaction buffer for DNase activity, including cofactors and the necessary buffer system to maintain pH in the reaction mixture, are introduced into tubes intended for real-time PCR reactions. The hydrolysis reaction of fluorescently labeled oligonucleotides is detected in real time in an appropriate PCR instrument at the optimum temperature for enzyme operation.

The technical result is also achieved by using a plasmid DNA substrate in the presence of an intercalating dye. A mixture containing a plasmid substrate that has been incubated with the EVA Green intercalating dye, an RNA-free sample, and a reaction buffer for DNase activity, including cofactors and the necessary buffer system to maintain pH in the reaction mixture. The plasmid DNA hydrolysis reaction is detected in real time in an appropriate PCR instrument at the optimum temperature for enzyme operation.

The technical result is also achieved by using the DNA substrate of oligonucleotides in the presence of an intercalating dye. An equilibrium mixture of a pair of oligonucleotides in a buffer containing 10 mM Tris-HCl pH 8.0, 25 mM NaCl is heated in a boiling water bath for 3 minutes. A mixture containing a DNA oligonucleotide substrate that has been incubated with the EVA Green intercalating dye, an RNA-free sample, and a reaction buffer for DNase activity, including cofactors and the necessary buffer system to maintain pH in the reaction mixture. The DNA hydrolysis reaction of the oligonucleotide substrate is detected in real time in an appropriate PCR instrument at the optimum temperature for enzyme operation.

The invention is illustrated by the following examples.

Example 1

Prepared a mixture of a pair of oligonucleotides presented in table 1 () 10 μm of each oligonucleotide in a buffer containing 10 mm Tris-HCl pH 8.0, 25 mm NaCl. This mixture was heated in a boiling water bath for 3 min, after which it cooled to room temperature.

Example 2

Prepared a mixture of a pair of oligonucleotides presented in table 2, 10 μm of each oligonucleotide in a buffer containing 10 mm Tris-HCl pH 8.0, 25 mm NaCl. This mixture was heated in a boiling water bath for 3 min, after which it cooled to room temperature.

Example 3

A mixture of 25 µL containing 7 µL of the substrate of fluorescently labeled oligonucleotides to a concentration of 2.8 µM, 1 µL of the test sample with the enzymatic preparation of DNase colicin E9 with concentrations in the reaction mixture of 0.8 µM, 0.4 μM, 0.2 μM, as well as 2.5 μl of the reaction buffer for work, including cofactors and the necessary buffer system to maintain pH in the reaction mixture, the concentration of the buffer in the reaction mixture (Tris-HCl 50 mm, NaCl 80 mm, MgCl ₂ 10 mm ). The hydrolysis reaction of fluorescently labeled oligonucleotides was detected in real time in an appropriate PCR instrument at a temperature of 30°C. The results are shown in figure 1: plot of fluorescence yield during incubation of fluorescently labeled oligonucleotides with 1-0.8 μm, 0.5-0.4 μm, 0.1-0.2 μm DNase. (ROX+Q) - fluorescently labeled oligonucleotides; (ROX+colicin) - DNase with fluorescently labeled oligonucleotide R-SD10 up (Rox).

Example 4

A 25 µl mixture containing 2.5 µl of pRSF-Duet-1 plasmid substrate was added to 100 µl thin-walled tubes to a final concentration in the reaction mixture of 8 nM, which was incubated for 30 minutes with 2.25 µM intercalating dye EVA Green, 1 µl of the test sample with the enzyme preparation of DNase colicin E9 with concentrations in the reaction mixture of 0.8 µM, 0.4 µM, 0.24 µM, as well as 2.5 µl of the reaction buffer for work, including cofactors and the necessary buffer system to maintain pH in reaction mixture buffer concentration in the reaction mixture (Tris-HCl 50 mm, NaCl 80 mm, MgCl ₂ 10 mm). The hydrolysis reaction of the plasmid substrate was detected in real time in an appropriate PCR instrument at a temperature of 30°C. The results are shown in figure 2: a graph of the fall in the yield of fluorescence depending on time when adding 0.1-0.08 μM of DNase to 200 ng of substrate, 0.3 * 2000.24 μM of DNase to 200 ng of substrate, 0.5 * 200- 0.4 μM DNase to 200 ng of substrate, (dye + colicin) - substrate with EVA Green; (plasmid without colicin) - EVA Green with DNase without DNA.

Example 5

A mixture of 25 µl containing 1 µl of oligonucleotide substrate DNA was added to thin-walled test tubes per 100 µl to a final concentration in the reaction mixture of 0.20 µM of the substrate, which was incubated for 30 minutes with 2.25 µM of the EVA Green intercalating dye, 1 µl of the test sample with the enzymatic preparation of DNase colicin E9 with concentrations in the reaction mixture of 0.8 μM, 0.32 μM, 1.6 μM, as well as 2.5 μl of the reaction buffer for work, including cofactors and the necessary buffer system to maintain pH in the reaction mixture concentration buffer in the reaction mixture (Tris-HCl 50 mM, NaCl 80 mM, MgCl2 10 mM). The hydrolysis reaction of the oligonucleotide substrate was detected in real time in an appropriate PCR instrument at a temperature of 30°C. The results are presented in figure 3: a graph of the fall of the yield of fluorescence depending on time at different amounts of DNase: 0.8; 0.32, 0.16 µM; DNase per 0.25 µM substrate. (K-) - substrate with intercalating dye; (K+) - intercalating dye with DNase.

Example 6

A 25 µl mixture containing 7 µl of a substrate of fluorescently labeled oligonucleotides to a concentration of 2.8 µM, 5 µl of a Staphylococcus aureus culture liquid sample, and 2.5 µl of reaction buffer for work, including cofactors and the necessary buffer system to maintain pH in the reaction mixture buffer concentration in the reaction mixture (Tris-HCl 50 mM, NaCl 10 mM, CaCl2 10 mM). The hydrolysis reaction of fluorescently labeled oligonucleotides was detected in real time in an appropriate PCR instrument at a temperature of 30°C. Results in figure 4: Plot of the fluorescence signal in samples with fluorescently labeled oligonucleotides: RQ - substrate in the presence of culture liquid St. aureus; (K-) - fluorescently labeled oligonucleotides with LB medium; (K+) - culture liquid St. aureus with fluorescently labeled oligonucleotide R-SD10 up (Rox).

Example 7

A mixture of 25 µl containing 2.5 µl of the plasmid pRSF-Duet-1 substrate was added to thin-walled tubes per 100 µl to a final concentration in the reaction mixture of 8 nM, which was incubated for 30 minutes with 2.25 µM of intercalating dye EVA Green, 5 µl of a sample of the cultural liquid of Staphylococcus aureus, which was incubated with RNase A until complete hydrolysis of the RNA present in the nutrient medium, as well as 2.5 µl of the reaction buffer for work, including cofactors and the necessary buffer system to maintain pH in the reaction mixture buffer concentration in the reaction mixture ( tris-HCl 50 mM, NaCl 10 mM, CaCl2 10 mM). The hydrolysis reaction of the plasmid substrate was detected in real time in an appropriate PCR instrument at a temperature of 30°C. Results in figure 5: Graph of the fluorescence signal in samples with 100 ng of pRSFDuet-1 plasmid DNA, EVA Green dye 2.25 cM in the presence of culture fluid: St. aureus and alkaline buffer; (O-) - the corresponding sample in the absence of a substrate; (K+) - the corresponding sample in the absence of substrate and intercalating dye; (K-) - substrate with intercalating dye and LB medium; (KC) - intercalating dye with LB medium; (C) - medium LB; (E) - intercalating dye.

Example 8

A mixture of 25 µl containing 1 µl of DNA of the oligonucleotide substrate was added to thin-walled tubes per 100 µl to a final concentration in the reaction mixture of 0.20 µM of the substrate, which was incubated for 30 minutes with 2.25 µM of the intercalating dye EVA Green, 5 µl of the culture sample. liquid of Staphylococcus aureus, which was incubated with RNase A until complete hydrolysis of the RNA present in the nutrient medium, as well as 2.5 μl of the reaction buffer for work, including cofactors and the necessary buffer system to maintain pH in the reaction mixture, the concentration of the buffer in the reaction mixture (Tris-HCl 50 mM, NaCl 10 mM, CaCl2 10 mM). The hydrolysis reaction of the oligonucleotide substrate was detected in real time in an appropriate PCR instrument at a temperature of 30°C. The results are shown in Figure 6: Plot of the fluorescence signal in samples with 2.8 μM oligonucleotides, 2.25 μM EVA Green dye in the presence of St. aureus and alkaline buffer; (O-) - the corresponding sample in the absence of a substrate; (K+) -corresponding sample in the absence of substrate and intercalating dye; (K-) - substrate with intercalating dye and LB medium; (KC) - intercalating dye with LB medium; (C) - medium LB;; (E) - intercalating dye.

Literature

1. Berends E.T.M. Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps // J. Innate Immun. 2010. T. 2. No. 6. C. 576-586.

2. E M. et al. Oligomer based real-time detection of microorganisms producing nuclease enzymes // Analyst. 2019. T. 144. No. 4. C. 1379-1385.

3. Gerceker D. et al. A new, simple, rapid test for detection of DNase activity of microorganisms: DNase Tube test // J. Gen. Appl. microbiol. 2009. T. 55. No. 4. C. 291-294.

4. JEFFRIES CD., HOLTMAN D.F., GUSE D.G. Rapid method for determining the activity of microorganisms on nucleic acids. // J. Bacteriol. 1957. T. 73. No. 4. pp. 590-591.

5. Schreier J.B. Modification of deoxyribonuclease test medium for rapid identification of Serratia marcescens. // Am. J.Clin. Pathol. 1969. T. 51. No. 6. C 711-716.

6. Detection of nuclease activity in semisolid and broth cultures - PubMed [Electronic resource]. URL: https://pubmed.ncbi.nlm.nih.gov/4185235/ (accessed 04/22/2021).

Claims (1)

A method for determining DNase activity, including the use of a substrate obtained by annealing two complementary oligonucleotides in the presence of the EVA Green intercalating dye, PCR in a mixture containing a DNA oligonucleotide substrate incubated with the EVA Green intercalating dye, an RNA-free sample, and a reaction buffer for the operation of DNase, including cofactors and a buffer system to maintain pH in the reaction mixture, the presence of non-specific DNase activity is determined by the real-time DNA hydrolysis reaction of the oligonucleotide substrate based on the drop in fluorescence in the test sample relative to the control sample.

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