RU2799795C1 - Method of amplification of short nucleic acid fragments by loop isothermal pcr - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of loop isothermal PCR is described. The proposed solution differs from the classical version of loop isothermal PCR (in the English version LAMP (loop-mediated isothermal amplification)) by one or more donor oligonucleotides introduced into the reaction, as well as primers specific for donor oligonucleotides.

EFFECT: invention expands the arsenal of tools for amplification of short DNA/RNA fragments, and invention can be used for analysis.

13 cl, 3 dwg, 5 tbl, 5 ex

Description

The invention relates to a variant of the loop isothermal PCR method. The proposed solution differs from the classical version of loop isothermal PCR (LAMP (loop-mediated isothermal amplification) in the English version) in that one or more donor oligonucleotides, as well as primers specific for donor oligonucleotides, are introduced into the reaction. The invention expands the arsenal of tools for amplification of short DNA/RNA fragments. The invention can be used for analysis.

State of the art

The main method for nucleic acid amplification is the polymerase chain reaction (PCR). However, in molecular diagnostics, there is a need for simpler, faster and cheaper methods of obtaining results than PCR.

There are a number of methods for isothermal amplification of nucleic acids. The most popular of them is loop isothermal amplification of DNA and RNA (LAMP and RT-LAMP) .

LAMP is a method of amplifying DNA at a constant temperature using a DNA polymerase capable of displacing the second strand of DNA and primers, upon completion of which amplicons containing hairpin structures are formed.

The protocol of the LAMP ( Loop -Mediated Isothermal Amplification ) method was developed by Japanese scientists more than 20 years ago [1]. The original protocol consists in the use of four oligonucleotide primers and a large Bst DNA polymerase fragment with strong displacement activity for DNA amplification. Four primers must be specific to 6 regions of the target fragment:
F3 - forward outer primer complementary to the F3c region; B3 - reverse outer primer complementary to the B3c region; FIP - forward internal primer consists of two parts: F1c (5') and F2 (3'), complementary sections of F1 and F2c, respectively; BIP - reverse internal primer consists of two parts: B1c (5') and B2 (3'), complementary plots B1 and B2c, respectively.

The process of amplification by the LAMP method can be divided into three main stages: 1) the formation of a basic dumbbell-shaped structure; 2) the stage of cyclic amplification; 3) the stage of elongation and repetition of cycles. As a result of such a reaction, many concatemeres of inverted repeats of the target fragment are formed, which form cauliflower- like structures in space. And if the length of the target region is usually 80-250 bp, then the length of the concatemers can reach 20 kb. and more.

Subsequently, the authors of the method proposed the use of 6 primers specific to 8 regions of the target fragment. Two more loop primers LoopF and LoopB should be complementary to the sequences between the F1 and F2 regions, as well as B1 and B2 of the target fragment. They come into play at the third stage of the reaction, annealing on single-stranded regions of previously formed DNA loops and becoming additional centers of polymerization initiation. In this case, RNA can also be used as a template; for this, reverse transcriptase must be added to the reaction mixture. This version of the method is called RT-LAMP ( reverse t ranscription - coupled LAMP ). More recently, New England Biolabs developed version 3.0 of Bst DNA polymerase, which has reversetase activity and eliminates the need for another enzyme in the reaction.

Selection of primers for LAMP is not a trivial task. And even there are recommendations to test more than one set of 4-6 oligonucleotides. But now special programs have been developed for automatic selection of primers for LAMP/RT-LAMP. One of the most popular programs is PrimerExplorer [2] or MorphoCatcher [3].

LAMP allows you to answer the question - was the target sequence present in the original sample (for example, pathogen DNA / RNA). To answer this question, it is enough to establish whether the products accumulated in the mixture during the reaction. To do this, upon completion of LAMP, the reaction mixture is stained with an intercalating dye (SYBR Green, ethidium bromide, propidium iodide, PicoGreen, etc.) and viewed under UV light. Moreover, it is possible to monitor the change in the fluorescence of the colored reaction mixture directly during the process using a real-time amplifier or a special real-time fluorimeter for LAMP.

It is possible to use not dyes, but various variants of fluorescently labeled FIP or BIP. In particular, FIP/BIP DARQ probes have been proposed for real-time multiplex LAMP/RT-LAMP [4].

Methods for detecting LAMP products are described in detail in articles by Becherer et al [5].

For effective LAMP amplification, less than 10 copies of the target DNA region in the initial reaction mixture is sufficient. At the same time, due to the lower sensitivity to impurities and inhibitors, even coarse lysates and biological fluids can be used as a sample.

Bst DNA polymerase (large fragment) - is a fragment of Bacillus stearothermophilus DNA polymerase with a molecular weight of 67 kDa. Enzyme activities: 5'→3' polymerase, 5'→3' displacement, weak revertase. This reagent was used by Japanese researchers in the development of the LAMP method.

Bst 2.0 DNA polymerase is an in silico developed version of Bst DNA polymerase, characterized by increased amplification rate and increased reaction yield. The enzyme is also characterized by greater thermal stability and salt concentration tolerance, which provides flexibility in reaction conditions.

Bst 2.0 WarmStart® DNA polymerase is a variant of Bst 2.0 DNA polymerase that, at temperatures below 45°C, is non-covalently bound to an aptamer that inhibits its activity. This allows the reaction mixture to be prepared at room temperature without fear of a premature start of polymerization.

Bst 3.0 DNA polymerase is a version of Bst DNA polymerase that, in addition to the advantages of Bst 2.0 DNA polymerase, has strong reversetase activity up to a temperature of 72°C. This allows you to set the reaction RT-LAMP without adding reverse transcriptase.

Many ready-made kits for LAMP and RT-LAMP have been developed, such as: WarmStart® loop isothermal amplification kit for DNA and RNA, ready-mix for colorimetric loop isothermal WarmStart® amplification of DNA and RNA.

The reaction also uses such enzymes as: WarmStart® RTx reverse transcriptase, Tte UvrD DNA helicase, Antarctic thermolabile uracil-DNA glycosylase.

The closest analogue of the proposed solution can be called RU 2673733 [6].

Described is a method for detecting a target nucleotide sequence of a toxigenic C. difficile in a sample, wherein said method includes bringing said sample into contact with at least one forward primer, at least one reverse primer, and at least one infiltrating oligonucleotide under conditions conducive to amplification of said target nucleotide sequence, wherein each said primer and said intruding oligonucleotide contains a site, complement ary of the specified target nucleotide sequence; wherein said intruder oligonucleotide converts at least a portion of the target nucleotide sequence to a single stranded state to allow binding of the forward primer and reverse primer, and said intruder oligonucleotide is an oligonucleotide greater than 30 nucleotides in length.

In the prior art there are no solutions related to methods of isothermal amplification of short fragments (any method of isothermal amplification, not only LAMP (loop), but also RCA, NASBA, RPA, etc.) using a donor oligonucleotide with the described parameters (length, structure) during amplification.

Donor oligonucleotides are commonly used in CRISPR-Cas technology [7], [8].

The essence of the invention

The main problem of the loop isothermal polymerase chain reaction (LAMP) is that the study requires a sufficiently extended region of the studied nucleic acid (at least 200 nucleotides). Due to the peculiarities of some nucleic acid sequences, such a site cannot be found, and researchers resort to numerous tricks: they make degenerate primers, make several sets of primers, and so on. This approach requires a lot of effort in the selection of conditions for the operation of the system, the rise in the cost of the system, etc.

The solution for this application allows:

1. Reduce the requirements for the sequence of the studied nucleic acid by shortening the studied sequence (from 10 or more nucleotides).

2. Simplify the primer selection procedure by reducing the number of primers to be selected.

3. Make part or all of the primers universal to significantly reduce the cost of the reaction.

4. Do not overload the developed LAMP-PCR system with an excess of primers, which simplifies the selection of system operating conditions.

The proposed solution differs from the classical version of loop isothermal PCR (LAMP (loop-mediated isothermal amplification) in English) in that one or more donor oligonucleotides are introduced into the reaction, as well as primers specific for donor oligonucleotides.

The donor oligonucleotide/s is/are 40 or more nucleotides in length (any reasonable length). The oligonucleotide is conditionally divided into 2 sections: the 3'-end (changeable) is complementary to the studied/desired nucleic acid sequence of the organism under study and the 5'-end (unchanged), which has no analogues with the nucleic acids of the studied organisms, in particular, has no analogues in any organisms. The 3'-complementary region is selected each time so that it is complementary to the desired sequence.

In the case of multiple donor nucleotides, the second donor nucleotide is structurally similar to the first, differing only in that the 3' end is fully complementary to the 3' end of the first donor oligonucleotide.

If two donor oligonucleotides are added to the reaction mixture, the primer pool includes primers for amplifying the unchanged parts of the donor oligonucleotides.

Analysis steps (version with two donor oligonucleotides):

The principle of analysis is based on the competitive binding of the introduced nucleic acid sample with donor oligonucleotides.

1. One of the donor oligonucleotides recognizes with its 3'-site the complementary site of the studied nucleic acid (DNA or RNA) and forms the complex "donor oligonucleotide - nucleic acid under study".

In the absence of the desired nucleic acid, the formation of the "donor oligonucleotide-test nucleic acid" complex does not occur.

2. The second donor nucleotide forms a complex with the "unbound" (free) first donor nucleotide in step 1.

3. The complex “first donor oligonucleotide - second donor oligonucleotide” is completed by polymerase: an amplicon is formed, which consists of the sequence of the first and second donor oligonucleotide.

4. In the presence of a set of primers, amplification of the amplicon formed at the second stage occurs.

Analysis steps (variant with one donor oligonucleotide):

1. The donor oligonucleotide recognizes with its 3'-site the complementary site of the studied nucleic acid (DNA or RNA) and forms the "donor oligonucleotide - the studied nucleic acid" complex.

In the absence of the desired nucleic acid, the formation of the "donor oligonucleotide-test nucleic acid" complex does not occur.

2. The complex "donor oligonucleotide - test nucleic acid" is completed by polymerase: an amplicon is formed, which consists of the sequences of the donor oligonucleotide and the test nucleic acid.

3. In the presence of a set of primers, amplification of the amplicon formed at the second stage occurs.

In FIG. 1 shows the principal structure of a donor oligonucleotide.

In FIG. 2 is a schematic diagram of the assay when two donor oligonucleotides are used.

In FIG. 3 is a schematic diagram of the assay when using a single donor oligonucleotide.

The solution presented in this application is a method for amplifying short nucleic acid fragments by loop isothermal PCR, including:

A) dilution of the first donor oligonucleotide;

In this case, a range of concentrations is possible, up to which a dilution of 1-1*10 ¹² pcs/µl is carried out. Preferably up to a concentration of 1*10 ⁷ pcs/µl.

B) dilution of the second donor oligonucleotide in several concentrations;

In this case, from 1 to an infinite number of points with concentrations of the donor oligonucleotide with dilutions in the range of 1-1*10 ¹² pieces/μl is possible. Preferably at points 1*10 ⁷ , 1*10 ⁶ , 1*10 ⁵ , 1*10 ⁴ pieces/µl.

C) sequential addition of 2 µl of water, 1 µl of water, 1 µl of the second donor oligonucleotide with different concentration points to each of the tubes, the number of tubes is determined by the formula: positive control tube + negative control tube + number of dilutions prepared at step B);

D) adding to all test tubes, except for the first one, 1 µl of the first donor nucleotide;

E) the mixture is heated at 45–100°C (the temperature depends on the annealing temperature of the complementary parts of donor oligonucleotides) for 2–15 minutes;

E) Amplification buffer, dNTP, primers, Bst polymerase, visualizing agent, for example, a fluorescent intercalating dye, are added to the obtained samples, if the method of detection by the accumulation of a fluorescent signal from an intercalating dye is used. It is possible to detect by changing the fluorescent signal using primers containing covalently “attached” fluorescent dyes in their structure. Turbodimetric detection is also possible, as well as visual detection by changing the color of the indicator that is added to the reaction mixture;

G) mix and place the samples in the device for amplification at 55-70 °C (the temperature depends on the calculated primer annealing temperature, on the one hand, and on the optimal temperature for enzyme operation. According to a specific embodiment, the calculated primer annealing temperature is 63-65 °C, the temperature optimum for enzyme operation is 58-70 °C, thus, the most preferable temperature is 65 °C.

Also claimed is a method for amplifying short fragments of nucleic acids by loop isothermal PCR, including:

A) dilute the first and second donor oligonucleotides to two selected concentrations; in this case, a concentration range of 1-1*10 ¹² pcs/µl is possible for each oligonucleotide. Preferably 1*10 ⁷ and 1*10 ⁴ pcs/µl.

B) sequentially added to several test tubes, the number of which is calculated by the formula: positive control + negative control + number of dilutions prepared at this stage, 2 µl of water, 1 µl of water, 1 µl of nucleic acid (RNA or DNA) in several concentrations;

In this case, from 1 to an infinite number of points with concentrations of the donor oligonucleotide with dilutions in the range of 1-1*10 ¹² pieces/μl is possible. Preferably at points 10 ⁴ pcs/µl, 10 ⁶ pcs/µl, 10 ⁸ pcs/µl.

C) add 1 µl of the second donor nucleotide to all tubes except the first one;

D) the mixture is heated at 45 - 100 ° C for 2-15 minutes;

E) 1 µl of the first donor nucleotide is added to all tubes and heated again at 45–100 °C for 2–15 minutes;

E) buffer for amplification, dNTP, primers, Bst polymerase, imaging agent are added to the obtained samples;

Preferably a fluorescent intercalating dye.

G) mix and place the samples in the device for amplification at 55 - 70 °C.

Also presented is a method for amplifying short nucleic acid fragments by loop isothermal PCR, including:

A) diluting the donor oligonucleotide; while a concentration range of 1-1*10 ¹² pcs/µl is possible for each oligonucleotide, preferably up to a concentration of 1*10 ⁷ pcs/µl.

B) in several tubes, the number of which is calculated by the formula: positive control + negative control + number of dilutions prepared at this stage, sequentially add 1 µl of water, 2 µl of water, 1 µl of nucleic acid (RNA or DNA) in several concentrations; In this case, a concentration range of 1-1*10 ¹² pcs/µl for nucleic acid (DNA or RNA) is possible. Preferably with a concentration of 10 ⁴ , 10 ⁶ , 10 ⁸

C) add 1 µl of the donor nucleotide to all tubes except the second one;

D) the mixture is heated at 45-100 °C for 2-15 minutes;

E) buffer for amplification, dNTP, primers, Bst polymerase, imaging agent are added to the obtained samples; preferably a fluorescent intercalating dye;

E) mix and place the samples in the device for amplification at 55-70 °C.

Detailed description of the invention

Example 1 : Detection of a complex formed by two donor oligonucleotides.

The first donor oligonucleotide is preliminarily diluted to a concentration of 1*10 ⁷ pcs/µl.

The second donor oligonucleotide is diluted at concentrations of 1*10 ⁷ , 1*10 ⁶ , 1*10 ⁵ , 1*10 ⁴ pcs/µl.

2 µl of water, 1 µl of water, 1 µl of the second donor oligonucleotide with a concentration of (10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ pcs/µl) were sequentially added to 6 test tubes. In all tubes, except for the first one, 1 µl of the first donor nucleotide was added. Thus, in test tube No. 1 - 2 µl of water, in test tube No. 2 - 1 µl of water + 1 µl of the first donor nucleotide, in test tube No. 3 - 1 µl of the first donor nucleotide + 1 µl of the second donor oligonucleotide with a concentration of 107 pcs/µl, in test tube No. 4 - 1 µl of the first donor nucleotide + 1 µl of the second donor oligonucleotide and with a concentration of 10 ^{6 pcs/µl, in tube No. 5 - 1 µl of the first donor nucleotide + 1 µl of the second donor oligonucleotide with a concentration of 10 5 pcs} /µl, in test tube No. 6 - 1 µl of the first donor nucleotide + 1 µl of the second donor oligonucleotide with a concentration of 10 ^{4 pcs} /µl.

The mixture was heated at 65°C for 5 minutes.

Amplification buffer, dNTP, primers, Bst polymerase, fluorescent intercalating dye were added to the obtained samples, mixed, and the samples were placed in the amplification device at 65 °C.

The results are presented in Table 1 and in FIG. 1.

Table 1. Isothermal PCR results. Sample name Tth, cycles*** Comments K (DO1)* 44.7 The amplification curve comes out late. K (DO2)** 54.9 The amplification curve comes out late. DO1 10 ⁷ : DO2 10 ⁷ 11.5 The amplification curve exits earlier than in K(DO1) and K(DO2), which means that the desired complex was formed. DO1 10 ⁷ : DO2 10 ⁶ 13.4 The amplification curve exits earlier than in K(DO1) and K(DO2), but later than in the previous line, which means that the complex was formed and its concentration is less than in DO1 10 ⁷ :DO2 10 ⁷ . DO1 10 ⁷ : DO2 10 ⁵ 15 The amplification curve exits early, but later than in the previous line, which means that the complex was formed and its concentration is less than in DO1 10 ⁷ :DO2 10 ⁶ . DO1 10 ⁷ : DO2 10 ⁴ 25 The amplification curve exits early, but later than in the previous line, which means that the complex was formed and its concentration is less than in DO1 10 ⁷ :DO2 10 ⁵ . * - control of a non-specific reaction that provokes the first donor nucleotide. ** - control of a non-specific reaction that provokes the second donor nucleotide. *** - 1 cycle = 30 seconds

Example 1 demonstrates the formation of a complex between donor oligonucleotides containing the sequence of interest, followed by detection of the formed complex. Moreover, the cycle size is inversely proportional to the concentration of the complex between the donor oligonucleotides, which indicates a specific reaction.

Example 2 : Nucleic acid detection using two donor oligonucleotides (Fig. 2) .

The first and second donor oligonucleotides are pre-diluted to a concentration of 1*10 ⁷ and 1*10 ⁴ pcs/µl, respectively.

Test RNA was pre-produced using a set of reagents (T7-tr-20). Calibration samples prepared. The function of the RNA is to form a complex with the donor oligonucleotide.

2 µl of water, 1 µl of water, 1 µl of RNA with a concentration (10 ⁴ pcs/µl, 10 ⁶ pcs/µl, 10 ⁸ pcs/µl) were sequentially added to 5 test tubes. In all tubes, except for the first one, 1 µl of the second donor nucleotide was added. In the first tube: 2 µl of water + 1 µl of the first donor nucleotide, in the tube 2: 1 µl of water + 1 µl of the second donor nucleotide+1 µl of the first donor nucleotide, in the tube 3: 1 µl of RNA with a concentration of 10 ⁴ pcs/µl + 1 µl of the second donor nucleotide + 1 µl of the first donor nucleotide, in the tube 4: 1 µl RNA with a concentration of 10 ⁶ pcs/µl + 1 µl of the second donor nucleotide + 1 µl of the first donor nucleotide, in a test tube 5: 1 µl of RNA with a concentration of 10 ⁸ pcs/µl + 1 µl of the second donor nucleotide + 1 µl of the first donor nucleotide.

The mixture was heated at 45°C for 15 minutes.

Next, 1 µl of the first donor nucleotide was added to all tubes and heated again at 65°C for 2 minutes.

Amplification buffer, dNTP, primers, 4 units of Bst polymerase, fluorescent intercalating dye were added to the obtained samples, mixed, and the samples were placed in the amplification device at 65 °C.

As a result, the following results were obtained:

Table 2. Sample name Tth, cycles** Comments K (DO1) * 61 The amplification curve comes out late. Without RNA 20.8 The amplification curve exits early, a complex is formed between the donor oligonucleotides. RNA 10 ⁸ pcs 65.9 When adding RNA containing the investigated fragment, there is competition between the second donor oligonucleotide and RNA for the first donor oligonucleotide. The concentration of the complex between the donor oligonucleotides decreases, and the cycle shifts upwards. RNA 10 ⁶ pcs 41.9 Similar to the previous one, but differs by a lower concentration of RNA> less competitor for the second donor oligonucleotide> more complex is formed between the donor oligonucleotides, therefore, the exit cycle of the curve is less than in the sample in "RNA 10 ⁸ pcs". RNA 10 ⁴ pcs 21.5 Similar to the previous one, but differs by a lower concentration of RNA> less competitor for the second donor oligonucleotide> more complex is formed between the donor oligonucleotides, therefore the exit cycle of the curve is less than in the sample in "RNA 10 ⁶ pcs". * - control of a non-specific reaction that provokes a donor nucleotide. ** - 1 cycle = 30 seconds

Example 3 : Nucleic acid detection using two donor oligonucleotides (Fig. 2) .

The first and second donor oligonucleotides are pre-diluted to a concentration of 1*10 ⁷ and 1*10 ⁴ pcs/µl, respectively.

DNA calibration samples were prepared.

2 µl of water, 1 µl of water, 1 µl of DNA with a concentration (10 ⁴ pcs/µl, 10 ⁶ pcs/µl, 10 ⁸ pcs/µl) were sequentially added to 5 test tubes. In all tubes, except for the first one, 1 µl of the second donor nucleotide was added. In the first tube: 2 µl of water + 1 µl of the first donor nucleotide, in the tube 2: 1 µl of water + 1 µl of the second donor nucleotide+1 µl of the first donor nucleotide, in the tube 3: 1 µl of DNA with a concentration of 10 ^{4 pcs/µl + 1 µl of the second donor nucleotide + 1 µl of the first donor nucleotide, in the tube 4 : 1 µl of DNA at a concentration of 10 6 pcs} /µl + 1 µl of the second donor nucleotide + 1 µl of the first donor nucleotide, in tube 5: 1 µl of DNA at a concentration of 10 ⁸ pcs/µl + 1 µl of the second donor nucleotide + 1 µl of the first donor nucleotide.

The mixture was heated at 100°C for 2 minutes.

Next, 1 µl of the first donor nucleotide was added to all tubes and heated again at 65°C for 2 minutes.

Amplification buffer, dNTP, primers, 4 units of Bst polymerase, fluorescent intercalating dye were added to the obtained samples, mixed, and the samples were placed in the amplification device at 65 °C.

As a result, the following results were obtained:

Table 3 Sample name Tth, cycles** Comments K (DO1) * 67 The amplification curve comes out late. Without DNA 19.8 The amplification curve exits early, a complex is formed between the donor oligonucleotides. DNA 10 ⁸ pcs 63.1 When adding DNA containing the fragment under study, there is competition between the second donor oligonucleotide and DNA for the first donor oligonucleotide. The concentration of the complex between the donor oligonucleotides decreases, and the cycle shifts upwards. DNA 10 ⁶ pcs 38.7 Similar to the previous one, but differs by a lower concentration of DNA> less competitor for the second donor oligonucleotide> more complex is formed between donor oligonucleotides, therefore, the exit cycle of the curve is less than in the sample in "DNA 10 ⁸ pcs". DNA 10 ⁴ pcs 21.1 Similar to the previous one, but differs by a lower concentration of DNA> less competitor for the second donor oligonucleotide> more complex is formed between donor oligonucleotides, therefore, the exit cycle of the curve is less than in the sample in "DNA 10 ⁶ pcs". * - control of a non-specific reaction that provokes a donor nucleotide. ** - 1 cycle = 30 seconds

Example 4 : Nucleic acid detection with a single donor oligonucleotide (FIG. 3) .

The donor oligonucleotide is preliminarily diluted to a concentration of 1*10 ⁷ pcs/µl.

Test RNA was preliminarily produced using a set of reagents (T7-tr-20, Biosan, Russia).

Calibration samples prepared. 1 µl of water, 2 µl of water, 1 µl of RNA with a concentration (10 ⁴ , 10 ⁶ , 10 ⁸ ) were sequentially added to 5 test tubes. In all tubes, except for the second one, 1 µl of the donor nucleotide was added. Thus, 1 μl of the donor nucleotide was added to all tubes, except for the second one.

The mixture was heated at 65°C for 2 minutes.

Amplification buffer, dNTP, primers, 4 units of Bst polymerase, fluorescent intercalating dye were added to the obtained samples, mixed, and the samples were placed in the amplification device at 65 °C.

As a result, the following results were obtained:

Table 4 Sample name Tth, cycles** Comments K (DO1) * - The result demonstrates that the DO1-RNA complex is not detected due to the absence of RNA. RNA 10 ⁸ pcs without DO1 - The result demonstrates that the DO1 and RNA complex is not detected due to the absence of DO1. RNA 10 ⁸ pcs 17.9 The result demonstrates that a complex of DO1 and RNA is detected. RNA 10 ⁶ pcs 26.4 The result demonstrates that a complex of DO1 and RNA is detected. The exit cycle is longer, since less RNA is introduced compared to the previous one, therefore, less RNA-DO1 complex is formed. RNA 10 ⁴ pcs 38.1 The result demonstrates that a complex of DO1 and RNA is detected. The exit cycle is longer, since less RNA is introduced compared to the previous one, therefore, less RNA-DO1 complex is formed. * - control of a non-specific reaction that provokes a donor nucleotide. ** - 1 cycle = 30 seconds

Example 5 : Nucleic acid detection using a single donor oligonucleotide (Figure 3) .

The donor oligonucleotide is preliminarily diluted to a concentration of 1*10 ⁷ pcs/µl.

DNA calibration samples were prepared. 1 µl of water, 2 µl of water, 1 µl of DNA with concentration (10 ⁴ , 10 ⁶ , 10 ⁸ ) were sequentially added to 5 tubes. In all tubes, except for the second one, 1 µl of the donor nucleotide was added. Thus, 1 μl of the donor nucleotide was added to all tubes, except for the second one.

The mixture was heated at 95°C for 2 minutes.

Amplification buffer, dNTP, primers, Bst polymerase, fluorescent intercalating dye were added to the obtained samples, mixed, and the samples were placed in the amplification device at 65 °C.

As a result, the following results were obtained:

Table 5 Sample name Tth, cycles** Comments K (DO1) * - The result demonstrates that the DO1-DNA complex is not detected due to the absence of DNA. DNA 10 ⁸ pcs without DO1 - The result demonstrates that the DO1-DNA complex is not detected due to the absence of DO1. DNA 10 ⁸ pcs 20.8 The result demonstrates that a complex of DO1 and DNA is detected. DNA 10 ⁶ pcs 28.3 The result demonstrates that a complex of DO1 and DNA is detected. The exit cycle is longer, since less DNA is introduced compared to the previous one, therefore, less DNA-DO1 complex is formed. DNA 10 ⁴ pcs 45.7 The result demonstrates that a complex of DO1 and DNA is detected. The exit cycle is longer, since less DNA is introduced compared to the previous one, therefore, less DNA-DO1 complex is formed. * - control of a non-specific reaction that provokes a donor nucleotide. ** - 1 cycle = 30 seconds

Thus, the above examples show that a complex of donor oligonucleotides containing a test fragment, or a complex of a nucleic acid containing a test fragment and a donor oligonucleotide, is formed and detected. The size of the studied sequence is 10 nucleotides or more, which for the proposed solution is approximately 20 times smaller than required for standard isothermal PCR. The size may be different. Actually achieved technical result is the reduction of requirements for the length of the fragment. In the examples given, the size of the investigated fragment of 19 nucleotides was also used.

Bibliography

1. Notomi T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000.28:E63. DOI: 10.1093/nar/28.12.e63.

2. http://primerexplorer.jp/e/

3. http://morphocatcher.ru/

4. Tanner N.A. et al. Simultaneous multiple target detection in real-time loop-mediated isothermal amplification. biotechniques. 2012.53:81-89. DOI: 10.2144/0000113902.

5. Becherer L. et al. Loop-mediated isothermal amplification (LAMP) - review and classification of methods for sequence-specific detection. Anal. methods. 2020. 12: 717-746. DOI: 10.1039/C9AY02246E.

6. RU 2673733 29.11.2018 C2.

7. Nucleic Acids Res. 2019 Nov 4;47(19): e116. doi:10.1093/nar/gkz669.

8. RU 2463350 10.10.2012 C2.

Claims (33)

1. A method for amplifying short fragments of nucleic acids by loop isothermal PCR, including: A) dilution of the first donor oligonucleotide; B) dilution of the second donor oligonucleotide in several concentrations; C) sequential addition of 2 µl of water, 1 µl of water, 1 µl of the second donor oligonucleotide with different concentration points to each of the tubes, the number of tubes is determined by the formula: positive control tube + negative control tube + number of dilutions prepared at step B); D) adding to all test tubes, except for the first one, 1 µl of the first donor nucleotide; E) the mixture is heated at 45-100°C for 2-15 minutes; E) buffer for amplification, dNTP, primers, Bst polymerase, imaging agent are added to the obtained samples; G) mix and place the samples in the device for amplification at 55-70°C. 2. The method according to claim 1, where the dilution in step A) is carried out to a concentration of 1×10 ⁷ pcs/µl. 3. The method according to claim 1, where at stage B) dilution is carried out with concentrations of the donor oligonucleotide at points 1⋅10 ⁷ , 1⋅10 ⁶ , 1⋅10 ⁵ , 1⋅10 ⁴ pieces/µl. 4. The method of claim 1, wherein in step E), the imaging agent is a fluorescent intercalating dye. 5. The method according to claim 1, where in stage G) the most preferred temperature is 65°C. 6. A method for amplifying short nucleic acid fragments by loop isothermal PCR, including: A) dilute the first and second donor oligonucleotides to two selected concentrations; B) sequentially added to several test tubes, the number of which is calculated by the formula: positive control + negative control + number of dilutions prepared at this stage; 2 µl of water, 1 µl of water, 1 µl of nucleic acid in several concentrations; C) add 1 µl of the second donor nucleotide to all tubes except the first one; D) the mixture is heated at 45-100°C for 2-15 minutes; E) 1 µl of the first donor nucleotide is added to all tubes and heated again at 45-100°C for 2-15 minutes; E) amplification buffer, dNTP, primers, Bst polymerase, reverse transcriptase, visualizing agent are added to the obtained samples; G) mix and place the samples in the device for amplification at 55-70°C. 7. The method according to claim 6, where in step A) the oligonucleotide is diluted at concentrations of 1⋅10 ⁷ and 1⋅10 ⁴ pcs/µl. 8. The method according to claim 6, where in step B) the donor oligonucleotide has dots with dilutions at concentrations of 10 ⁴ pcs/µl, 10 ⁶ pcs/µl, 10 ⁸ pcs/µl. 9. The method of claim 6 wherein in step E) the imaging agent is a fluorescent intercalating dye. 10. A method for amplifying short nucleic acid fragments by loop isothermal PCR, including: A) diluting the donor oligonucleotide; B) in several test tubes, the number of which is calculated by the formula: positive control + negative control + number of dilutions prepared at this stage; sequentially add 1 µl of water, 2 µl of water, 1 µl of nucleic acid in several concentrations; C) add 1 µl of the donor nucleotide to all tubes except the second one; D) the mixture is heated at 45-100°C for 2-15 minutes; E) amplification buffer, dNTP, primers, Bst polymerase, reverse transcriptase, visualizing agent are added to the obtained samples; E) mix and place the samples in the device for amplification at 55-70°C. 11. The method according to claim 10, where in step A) the oligonucleotide is diluted to a concentration of 1×10 ⁷ pcs/µl. 12. The method according to claim 10, where at stage B) dilutions are carried out with a concentration of 10 ⁴ , 10 ⁶ , 10 ⁸ . 13. The method of claim 10 wherein the imaging agent is a fluorescent intercalating dye.