RU2644262C1 - Method of detection of a set of nucleotide sequences in a sample by the method of coding amplicones in the containing polymerase chain reaction - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to the field of biotechnology, particularly to a method of simultaneous detection of multiple nucleotide sequences in a single polymerase chain reaction.

EFFECT: proposed solution allows to perform amplification of a plurality of nucleotide sequences in one sample in a single reaction by coding them with the ratio of a specific structure forward primers number.

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Description

FIELD OF THE INVENTION

The invention relates to the field of biotechnology, and in particular to a method for the simultaneous detection of several specific nucleotide sequences and nucleotide substitutions using multiplex polymerase chain reaction (PCR), and can be used to identify various pathogens of diseases, genotyping of humans and animals, detection of genetically modified organisms, etc. .P.

State of the art

Several methods are known for detecting multiple nucleotide sequences in a single sample. The most widely used today is xMAP (Reslova N., Michna V., Kasny M., Mikel P. and Kralik P. (2017) xMAP Technology: Applications in Detection of Pathogens. Front. Microbiol. 8:55. Doi: 10.3389 / fmicb.2017.0005555). Its essence is as follows. The PCR reaction product is encoded using the color of the carrier to which it is attached. As a carrier (solid phase), polystyrene microspheres d = 5.6 μm are used. Two fluorophores in various concentrations are integrated into the composition of the microspheres. A certain concentration ratio of fluorophores creates 100 possible types of particles, each of which will have its own unique spectral characteristic. The spectrum of a particle is its individual "number" and determines its type (variant). Particle type is recognized by the instrument (e.g. Luminex 100/200) during the measurement process. Various types of biological molecules can be fixed on the polystyrene surface, including fragments of oligonucleotide sequences complementary to those detected in the sample. Prior to hybridization with polystyrene-fixed oligonucleotides, the detected sequences are labeled with fluorescent labels. Recognizing the microsphere and determining the fluorescence of the sequence attached to it, we can understand which nucleotide sequence was found in the sample. The disadvantages of this method include the need to produce special microspheres for the analysis and use of individual labeled oligonucleotides for each detected sequence.

In (Huang Q, Zheng L, Zhu Y, Zhang J, Wen H, et al. (2011) Multicolor Combinatorial Probe Coding for Real-Time PCR. PLoS ONE 6 (1): e16033. Doi: 10.1371 / journal.pone .0016033) a method is proposed for coding real-time PCR products using combinations of replacement probes of different colors for one target sequence, complementary to one common sequence with a quencher. When a detectable sequence is detected in the reaction mixture, a combination of a color signal is recorded, by which the result of the reaction is determined. However, the disadvantages of this technology should be recognized as low multiplexability (up to 15 variants, according to the authors) and the need to synthesize several labeled oligonucleotides for one detectable sequence.

There are technologies for introducing nucleotide sequences into amplicons that allow different amplicons to be labeled with the same type of labeled oligonucleotides (Whitcombe D., Brownie J., Gillard H., McKechnie D., Theaker J., Newton C. and Little S. (1998) A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping Clinical Chemistry 44: 5, p. 918-923; Zhang Y., Zhang D., Li W., Chen J., Peng Y. and Cao W. (2003) A novel real-time quantitative PCR method using attached universal template probe Nucleic Acids Research, 31:20, e. 123 .; Rickert A., Lehrach H. and Sperling S. (2004) Multiplexed Real-Time PCR Using Universal Reporters. Clinical Chemistry 50: 9, p. 1680-1683). Typically, these are nucleotide sequences used as TaqMan probes for real-time PCR. The use of these methods for multiplexing PCR is limited by the number of possible channels for detecting fluorescent labels, which today does not exceed six.

Thus, the development of new methods for the separate determination of multiple nucleotide sequences in one sample is relevant for molecular biology. This is especially important for genotyping, when it is required to identify the presence of multiple mutations in a single sample in the form of single nucleotide substitutions, deletions, insertions, etc.

Disclosure of invention

The objective of the invention is to develop a new method for the individual identification of nucleotide sequences amplified during multiplex PCR, which can significantly increase the number of sequences detected during a single reaction, which in turn will significantly reduce the time required to complete the study and reduce its cost. The application of the proposed method will expand the scope of the use of genotyping methods in humans and animals for both research and diagnostic purposes.

The technical result of the invention is achieved by the fact that a new method of preparing the reaction mixture during PCR is proposed, the use of which allows to obtain several variants of amplicons in a given ratio from one detectable nucleotide sequence, which can then be detected by concentration on a separate surface area and determine from the ratio which sequence nucleotides amplified during the reaction.

A brief description of the drawings and their materials

In FIG. 1 shows the oligonucleotides used in example 1 for the detection of a single sequence.

In FIG. 2 shows the results of the assessment of the fluorescence of dyes in different ratios.

In FIG. Figure 3 shows the oligonucleotides used in Example 2 to detect a single nucleotide substitution in a nucleotide sequence.

In FIG. Figure 4 shows the results of the assessment of the fluorescence of dyes, which characterize the amplified nucleotide sequence by their ratio.

In FIG. 5 shows the oligonucleotides used in example 3 for independent detection of two nucleotide sequences in one sample.

In FIG. 6, the results of the assessment of the fluorescence of the dyes are presented when one of the detected sequences was in the sample.

In FIG. 7 shows the results of the assessment of the fluorescence of dyes when both nucleotide sequences were in the sample.

In FIG. 8 is a drawing of a method for encoding PCR products by attaching labels to them in a predetermined ratio.

The essence of the method is that during PCR, each detected nucleotide sequence, if present in the sample, is encoded in a certain way. This then allows using this code to determine its presence in the sample, regardless of the presence of other nucleotide sequences.

The coding of the nucleotide sequence determined by the PCR method in the sample is carried out by obtaining a certain ratio of amplification products that differ in different labels or regions for hybridization of oligonucleotides labeled with different labels.

The essence of the method is illustrated in Figure 8. A certain ratio of amplification products is obtained at the amplification stage by introducing several direct primers (1, 2) into the reaction mixture in the same ratio. Direct primers contain two sections. One region located at the 3 'end of the primer is the same for all direct primers and is complementary (or partially complementary) to the target oligonucleotide sequence in sample (3). The second section, located at the 5 'end of the primer, is different for each type of direct primer. This region is not complementary (or partially complementary) to the target sequence in the sample, has an identifiable label, or contains a template for an oligonucleotide sequence complementary to one of the labeled oligonucleotides. The ratio of the number of direct primers carrying templates for a sequence complementary to one of the labeled oligonucleotides, after completion of the cyclic amplification process, will accurately determine the ratio of the number of obtained amplicons with labeled regions or complementary to oligonucleotides labeled with various labels.

In the process of encoding the amplification product, one reverse primer (4) is used that is complementary (or partially complementary) to the portion of the nucleotide chain on the test sample located at a small distance from the attachment site of the forward primer (5).

After amplification is performed, the amplification products (6) are hybridized with oligonucleotides labeled with different labels (7), if there were no labels in the direct primers.

To recognize the code determined by PCR in the sample of the nucleotide sequence, hybridization of the labeled amplification products with oligonucleotides (8) complementary to the nucleotide sequence in the amplification products and fixed on some surface (9) at a minimum distance from each other is used.

The result is a surface area with a complex of oligonucleotides attached to it with different labels in a ratio determined by the ratio of direct primers introduced into the reaction mixture. Hybridization with attached oligonucleotides can be carried out after hybridization of amplification products with different labeled oligonucleotides, during hybridization of amplification products with different labeled oligonucleotides or after hybridization of amplification products with different labeled oligonucleotides. It is possible to carry out hybridization with oligonucleotides fully or partially complementary to amplicons before fixing them on a surface site, after which a complex of oligonucleotides is already fixed.

Evaluation of the ratio of the presence of different labels on the oligonucleotides allows us to determine the ratio of direct primers in the reaction mixture at the amplification stage and to identify the nucleotide sequence encoded by the ratio of the number of amplification products.

To identify two or more nucleotide sequences in one sample, each of them is encoded according to the method described above. For code recognition, oligonucleotides that are complementary (or partially complementary) to different amplicons are fixed on different non-intersecting parts of the surface. Assessment of the presence and number of tags is carried out individually at each such site. A different ratio of the number of different labels on the oligonucleotides at different sites will accurately determine the presence or absence of amplification products of a portion of the nucleotide chain in the test sample.

Labels used to identify oligonucleotides, preferably detected in the optical range of electromagnetic waves. Any other tags identified by their electromagnetic, chemical or any other properties may be used.

The implementation of the invention

The overall effectiveness of the proposed method for detecting multiple nucleotide sequences during one PCR was evaluated. Examples of specific implementation of the detection method for the detection of different nucleotide sequences and single nucleotide substitutions.

Example 1

The method is as follows to detect the presence of a specific nucleotide sequence in the sample:

1. A set of primers is synthesized, labeled oligonucleotides, biotinylated oligonucleotides, test samples of oligonucleotides (table 1). Used oligonucleotides synthesized by Synthol and Beagle. Purified by polyacrylamide gel electrophoresis. They were delivered in a lyophilized form, before use they were bred according to the attached instructions.

2. The reaction mixture for PCR is prepared, in which direct primers (total amount 1 μM) are added in the ratio of (Direct primer 1): (Direct primer 2) equal to 1: 2 and the reverse primer (1 μM). The mixture is prepared in two test tubes, in one of which (No. 1) a test sample (0.1 μM) is added.

3. Amplification is performed on the Tercik amplifier in the following mode:

1. Initiation of denaturation 94 ° C - 1 minute.

2. Denaturation 94 ° C - 30 sec.

3. Annealing of primers 37 ° C - 30 sec.

4. Elongation 72 ° С - 30 sec.

5. Repeat paragraphs 2-4 30 times.

6. Denaturation 94 ° C - 2 minutes.

7. Cooling to room temperature.

4. Amplification products are hybridized with labeled oligonucleotides 1 and 2. To this end, labeled oligonucleotides 1 and 2 in an amount of 2 μM were added to the reaction mixture, a denaturation cycle was carried out at 94 ° C for 30 seconds and slow cooling to room temperature over 30 minutes .

5. On streptavidin-coated magnetic balls (5 × 10 ⁵ pieces), biotinylated oligonucleotides (2 μM) are attached at a temperature of 37 ° C for 30 minutes. After that, the balls are washed three times with phosphate buffer using a magnetic tripod.

6. Hybridization of amplicons with oligonucleotides on magnetic balls (5 × 10 ⁵ pieces) is carried out for 1 hour at room temperature with continuous stirring. After that, the balls are washed three times with phosphate buffer to remove unreacted components using a magnetic tripod.

7. The balls are suspended in a buffer solution, placed in a Goryaev chamber and photographed using an Olympus BX-41 fluorescence microscope (Japan). Fluorescence is excited using a fluorescence filter cube to excite blue light BP470-490 / DM500 / BA515. 10 photos of random fields of view are taken. Used lens x100 Plan semi Apochromat N, NA = 1.3, WD = 0.2, CC = 0.17. Non-fluorescent oil is used for immersion. Shooting is made on the Olympus E-330 camera (Japan) in a resolution of 7.5 megapixels. JPEG image format.

8. On each photo, using computer microtelemetry in the VideoTest Morpho 5.0 program (Russia), the luminescence intensity in the FAM and HEX dye spectra of randomly selected balls is determined in the amount of 10 pieces (total number of measurements in each spectrum is 100). The results are shown in table 2.

9. Thus, as a result of applying the method of coding the amplification reaction product, it was found that the ratio of the intensity of the luminescence of the dyes on the labeled oligonucleotides attached to a magnetic ball corresponds to the ratio of direct primers introduced into the reaction mixture.

Example 2

The method is as follows to detect the presence of one single nucleotide substitution in the sample:

1. A set of primers is synthesized, labeled oligonucleotides, biotinylated oligonucleotides, test samples of oligonucleotides (table 3). Used oligonucleotides synthesized by Synthol and Beagle. Purified by polyacrylamide gel. They were delivered in a lyophilized form, before use they were bred according to the attached instructions. Primers 1.1 and 1.2 have at the 3 'end one non-complementary base (C) and one complementary base (A). Primers 2.1 and 2.2 have two non-complementary bases at the 3 'end (CC). Primers 1.1 and 1.2 have the ability to amplify (the last base is complementary to the sample), primers 2.1 and 2.2 are not able to amplify when there is a replacement (T> G) (the last bases are not complementary to the sample). In the absence of substitution (in sample 2, base G), amplification by a second group of direct primers is possible.

2. Three tubes are prepared with the reaction mixture for PCR to which direct primers are added (total amount 1 μM) in the ratio (Direct primer 1.1): (Direct primer 1.2) equal to 1: 2, direct primers (total amount 1 μM) in the ratio (Forward Primer 2.1): (Forward Primer 2.2) equal to 2: 1 and reverse primer (1 μM). Test sample 1 (0.1 μM) is added to test tube No. 1, test sample 2 (0.1 μM) is added to test tube No. 2, test samples 1 and 2 (0.1 μM each) are added.

3. Amplification is performed on the Tercik amplifier according to the following protocol:

1. Initiation of denaturation 94 ° C - 1 minute.

2. Denaturation 94 ° C - 30 sec.

3. Annealing of primers 37 ° C - 30 sec.

4. Elongation 72 ° С - 30 sec.

5. Repeat paragraphs 2-4 30 times.

6. Denaturation 94 ° C - 2 minutes.

7. Cooling to room temperature.

4. Amplification products are hybridized with labeled oligonucleotides 1 and 2. To this end, labeled oligonucleotides 1 and 2 in an amount of 2 μM were added to the reaction mixture, a denaturation cycle was carried out at 94 ° C for 30 seconds and slow cooling to room temperature over 30 minutes .

5. On streptavidin-coated magnetic balls (15 × 10 ⁵ pieces) biotinylated oligonucleotides (6 μM) are attached at a temperature of 37 ° C for 30 minutes. After that, the balls are washed three times with phosphate buffer using a magnetic tripod.

6. Hybridization of amplicons with oligonucleotides on magnetic balls (5 × 10 ⁵ pieces) is carried out for 1 hour at room temperature with continuous stirring. After that, the balls are washed three times with phosphate buffer to remove unreacted components using a magnetic tripod.

7. The balls are suspended in a buffer solution, placed in a Goryaev chamber and photographed using an Olympus BX-41 fluorescence microscope (Japan). Fluorescence is excited using a fluorescence filter cube to excite blue light BP470-490 / DM500 / BA515. 10 photos of random fields of view are taken. Used lens x100 Plan semi Apochromat N, NA = 1.3, WD = 0.2, CC = 0.17. Non-fluorescent oil is used for immersion. Shooting is made on the Olympus E-330 camera (Japan) in a resolution of 7.5 megapixels. JPEG image format.

8. On each photo, using computer microtelemetry in the VideoTest Morpho 5.0 program (Russia), the luminescence intensity in the FAM and HEX dye spectra of randomly selected balls is determined in the amount of 10 pieces (total number of measurements in each spectrum is 100). Only balls with a glow are counted. The results are shown in table 4.

9. Thus, as a result of applying the method of coding the amplification reaction product to detect single nucleotide substitutions, it was found that the ratio of the intensity of the luminescence of dyes on labeled oligonucleotides attached to a magnetic ball corresponds to the ratio of direct primers introduced into the reaction mixture. If both variants of substitution are present in the sample (conditional heterozygote), the signal is summed from both groups of primers, which confirms the possibility of using the proposed coding method for genotyping by single nucleotide substitutions.

Example 3

The method is as follows to detect the presence of two different sequences in the sample:

1. A set of primers is synthesized, labeled oligonucleotides, biotinylated oligonucleotides, test samples of oligonucleotides (table 5). Used oligonucleotides synthesized by Synthol and Beagle. Purified by polyacrylamide gel. They were delivered in a lyophilized form, before use they were bred according to the attached instructions. Primers 1.1 and 1.2 are used to detect the sequence of test sample 1. Primers 2.1 and 2.2 are used to detect the sequence of test sample 2.

2. Three tubes are prepared with the reaction mixture for PCR to which direct primers are added (total amount 1 μM) in the ratio (Direct primer 1.1): (Direct primer 1.2) equal to 1: 2, direct primers (total amount 1 μM) in the ratio (Forward Primer 2.1): (Forward Primer 2.2) is 2: 1 and reverse primers 1 and 2 (1 μM each). Test sample 1 (0.1 μM) is added to test tube No. 1, test sample 2 (0.1 μM) is added to test tube No. 2, test samples 1 and 2 (0.1 μM each) are added.

3. Amplification is performed on the Tercik amplifier according to the following protocol:

1. Initiation of denaturation 94 ° C - 1 minute.

2. Denaturation 94 ° C - 30 sec.

3. Annealing of primers 37 ° C - 30 sec.

4. Elongation 72 ° С - 30 sec.

5. Repeat paragraphs 2-4 30 times.

6. Denaturation 94 ° C - 2 minutes.

7. Cooling to room temperature.

4. Amplification products are hybridized with labeled oligonucleotides 1 and 2. To this end, labeled oligonucleotides 1 and 2 in an amount of 2 μM were added to the reaction mixture, a denaturation cycle was carried out at 94 ° C for 30 seconds and slow cooling to room temperature over 30 minutes .

5. streptavidin-coated magnetic beads on May ¹⁰ × 15 pieces in two tubes # 1 and # 2 is performed attaching biotinylated oligonucleotide 1 (# 1 in vitro) and 2 (# 2 in vitro) of 6 M at 37 ° C for 30 minutes. After that, the balls are washed three times with phosphate buffer using a magnetic tripod.

6. 5 × 10 ⁵ pieces of magnetic beads with biotinylated oligonucleotides from tubes # 1 and # 2 are added to each tube with amplification products. Amplicons are hybridized with oligonucleotides on magnetic beads for 1 hour at room temperature with continuous stirring. After that, the balls are washed three times with phosphate buffer to remove unreacted components using a magnetic tripod.

7. The balls are suspended in a buffer solution, placed in a Goryaev chamber and photographed using an Olympus BX-41 fluorescence microscope (Japan). Fluorescence is excited using a fluorescence filter cube to excite blue light BP470-490 / DM500 / BA515. 10 photos of random fields of view are taken. Used lens × 100 Plan semi Apochromat N, NA = 1.3, WD = 0.2, CC = 0.17. Non-fluorescent oil is used for immersion. Shooting is made on the Olympus E-330 camera (Japan) in a resolution of 7.5 megapixels. JPEG image format.

8. On each photo, using computer microtelemetry in the VideoTest Morpho 5.0 program (Russia), the luminescence intensity in the FAM and HEX dye spectra of randomly selected balls is determined in the amount of 10 pieces (total number of measurements in each spectrum is 100). Only balls with a glow are counted. The results are shown in table 6.

9. In test tube No. 3, the presence of a glow of two types of balls was detected. The first type included FAM and HEX signals in the ratio 1: 2, the second type 2: 1. We calculated the number of balls of two types of glow in a group of 100 randomly selected balls, the results are presented in table 7.

10. Thus, as a result of applying the method of encoding the amplification reaction product to detect two different sequences, it was found that the ratio of the intensity of the luminescence of the dyes on the labeled oligonucleotides attached to a magnetic ball corresponds to the ratio of direct primers introduced into the reaction mixture. If both variants of the sequence are present in the sample, the appearance of separate carriers with different color codes in the aggregate of detecting carriers (balls) can be used to determine which sequence is present in the sample.

Claims (5)

1. A method for detecting several specific nucleotide sequences in one sample by PCR during one reaction, comprising preparing a reaction mixture, characterized in that direct primers of several types with different labels are added to it, encoding the type of detectable ratio of their amount sequences, and one type of reverse primer for each detectable sequence, amplification, hybridization of the obtained amplicons with closing captured on different parts of the surface by oligonucleotides, where each individual part of the surface contains oligonucleotides that are complementary only to amplicons from one detectable sequence, reading the signal from labels on amplicons located on one surface area, and identifying different detected nucleotide sequences by the ratio of the signals of different labels, which repeats the ratio of the amounts of direct primers labeled with different labels in the initial reaction mixture. 2. The method according to p. 1, characterized in that the direct primers have a plot that is not complementary (or partially complementary) to the detected sequence, which contains a nucleotide sequence that is complementary (or partially complementary) to oligonucleotides with labels that hybridize with amplicons after amplification and, thus, each amplicon is labeled according to the direct primer built into it. 3. The method according to p. 1, characterized in that on a separate surface area nucleotide sequences are fixed, partially complementary to amplicons. 4. The method according to p. 1, characterized in that the hybridization of amplicons with oligonucleotides is first carried out, which are then attached to a specific surface area. 5. The method according to p. 1, characterized in that an oligonucleotide is attached to the surface, having a complementary (or partially complementary) sequence to the site of the additional oligonucleotide, another part of which has a complementary (or partially complementary) sequence to the amplification product (amplicon).

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