RU2415932C1 - Method for identifying extended spectrum class a beta-lactamase genes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: gene and point mutations therein are identified by hybridisation of DNA biotin marked within polymerase chain reaction, with microchip immobilised specific oligonucleotides. Biotin in microchip DNA duplexes is detected by a streptavidin-peroxidase conjugate that is followed with colorimetric peroxidase detection.

EFFECT: invention can be used in clinical microbiological laboratories and epidemiological surveillance services for assessing resistance of an agent of infection of Enterobacteriaceae family to antibiotics of penicillin and cephalosporin group by observing the mutations in coding genes.

5 dwg, 2 tbl, 4 ex

Description

The invention relates to the field of molecular biology, chemical enzymology and analytical biotechnology and can be used in clinical microbiological laboratories and epidemiological control services to detect the resistance of pathogens of the Enterobacteriaceae family to antibiotics of the penicillin and cephalosporins group by the presence of beta-lactamase genes and mutations in them. The invention is a method for conducting hybridization analysis on a DNA microchip with specific immobilized oligonucleotides for the simultaneous detection of three types of class A beta-lactamases genes (TEM, SHV and CTX-M) and point mutations in them, leading to a change in the substrate specificity of these enzymes.

Beta-lactam antibiotics account for more than half of all currently used antibiotics, among which the penicillins and cephalosporins are most widely used, especially in the treatment of a wide range of severe infections caused by gram-negative microorganisms of the Enterobacteriaceae family. The main factor limiting their clinical effectiveness is the ability of microorganisms to develop resistance to their action - antibiotic resistance. Several mechanisms of resistance of microorganisms to penicillins and cephalosporins are known, but the main of them is enzymatic hydrolysis with beta-lactamase enzymes formed in the cell wall [Paterson D.L. et al. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev., 2005, 18, pp. 657-686]. Beta-lactamases represent an extensive group of genetically and functionally different enzymes, characterized by the ability to destroy the beta-lactam ring, as a result of which the antibiotic loses its antimicrobial activity. Of the diversity of beta-lactamases, the greatest threat is extended-spectrum beta-lactamases (BLRS), which cause resistance in the initially sensitive microorganisms of the Enterobacteriaceae family [Perez F. et al. The continuing challenge of ESBLs. Curr Opin Pharmacol. 2000, 7, 459-469]. Due to plasmid localization of genes, the distribution of these enzymes among pathogens of human infectious diseases, primarily infections, is increasing every year. In certain medical institutions of the Russian Federation, the frequency of BLRS production among some microorganisms exceeds 90%. The most famous and common types of BLRS are TEM, SHV and CTX-M.

All described TEM type enzymes are variants of TEM-1 beta-lactamase and differ from the initial enzyme by single amino acid substitutions (from one to seven). The key to expanding the spectrum of substrate specificity are mutations at positions 104, 164, 238, and 240.

SHV type enzymes are derivatives of SHV-1 beta-lactamase and differ from it by the presence of point mutations. The key to changing the profile of substrate specificity are mutations at positions 35, 238, and 240.

Recently, in many countries of the world, including the Russian Federation, there has been a rapid spread of STX-M type BLRS [Edelstein M., Pimkin M., Palagin I., Edelstein I., Stratchounski L. Prevalence and molecular epidemiology of CTX- M extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian hospitals. Antimicrob. Agents Chemother. 2003, 47, 3724-3732]. Based on the homology of amino acid sequences, these enzymes are divided into 4 phylogenetic groups (subclusters). The expansion of the spectrum of their substrate specificity is also associated with the occurrence of single mutations. The key mutations that determine the spectrum of substrate specificity for cephalosporins are mutations at positions 167 and 240. Mutations at positions 26, 230, 253, 278 for the CTX-M-2 subcluster, and positions 121, 231 for the CTX-M-9 subcluster are important for recognition of CTX-M type enzymes common in various countries, including Russia.

Standard clinical methods for determining the nature of pathogens that cause infectious diseases and determining antibiotic resistance are based on the phenotypic characterization of pathogens. These methods are quite long in time; the interpretation of the data on the sensitivity of strains to antibiotics can be ambiguous. Traditional microbiological methods for detecting BLRS at best make it possible to assess the fact of the presence of an enzyme, but they cannot provide information about which of the enzymes is present, while none of the traditional microbiological methods can detect BLRS in 100% of strains. The sensitivity criteria of gram-negative microorganisms to third generation cephalosporins were determined by the National Committee for Clinical Laboratory Standards (NCCLS) only for E. coli and Klebsiella spp. At the same time, BLRS production is described in almost all representatives of the Enterobacteriaceae family and a number of other gram-negative microorganisms.

Molecular genetic methods make it possible to more accurately and fully identify and characterize the genes responsible for the development of resistance. A quick and economical method for detecting BLRS is to amplify target genes in PCR. The method of multi-primer PCR to identify the genes of beta-lactamases SHV, TEM and CTX-M types [Monstein H.J. et al. Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blАТХ-M genes in Enterobacteriaceae. APMIS, 2007, 115, 1400-1408]. As a result of multi-primer PCR, fragments of the SHV, TEM, and CTX-M types of beta-lactamases are amplified, based on the size of which gene type is identified. It is not possible to differentiate genes within these groups by this method, and further study of the genes is necessary in order to identify possible mutations that determine the extended spectrum of activity.

A promising method for identifying genes is microarray genotyping methods. Technologies developed using biological microchips are currently being actively developed in the world to identify and identify biological material [Heller M.J. DNA microarray technology: devices, systems, and applications. Annu Rev Biomed Eng. 2002, 4, 129-53]. DNA microarrays are a substrate of glass, silicon, porous membrane materials, and polymeric carriers onto which DNA fragments, synthetic oligonucleotides, proteins, or other probe molecules that can exhibit biological activity are applied in a matrix. DNA microarrays for identifying genes and mutations in them are based on hybridization of the studied nucleotide sequence with known oligonucleotides or DNA sequences immobilized on the surface of the microarray arranged in a specific order. The technology of DNA chips allows for multi-parameter analysis and identification of the pathogen and its resistance to antibiotics at the molecular level. This technology is more informative and has significant advantages over traditional molecular biological methods, because it allows miniaturization of the test sample and analyzer, which significantly reduces the cost of analysis and the time it takes, as well as simultaneously determine the various parameters of the test sample.

A DNA macrochip based on a membrane carrier is described for identification of STX-M type beta-lactamase genes and individual mutations in them by hybridization analysis [Ensor V.M. et al. A novel reverse-line hybridization assay for identifying genotypes of CTX-M-type extended-spectrum β-lactamases. J. Antimicrob. Chemotherapy, 2007, 59, 387-395]. The disadvantage of this approach is the “macro” format of the chip, fragments of the CTX-M type beta-lactamases are amplified during the polymerase chain reaction, as a result, it is possible to determine only a limited number of mutations. The conditions for determining mutations in genes belonging to different subclusters of CTX-M type beta-lactamases are different, therefore, the determination is carried out on different types of chips, which complicates the use of the method in epidemiological studies.

Closest to the claimed is a method of hybridization analysis on DNA microarrays with fluorescence detection to identify the TEM type beta-lactamase gene and mutations in them [European Patent 1580281 A2, date of publication September 28, 2005, Bulletin 2005/39]. In this method, the selection of sequences of oligonucleotides identical to the structure of the TEM type beta-lactamase gene was carried out and they were immobilized on the surface of glass microarrays. The method of hybridization analysis included the following stages: extraction of DNA from a sample, amplification of a specific TEM type beta-lactamase gene with simultaneous introduction of a Cy5 fluorescent label, DNA fragmentation, hybridization of labeled DNA with immobilized oligonucleotides, detection of hybridization results by activity of the fluorescent label.

The objective of the invention is to develop a method for the simultaneous highly specific determination of the main types of beta-lactamase class A genes (TEM, SHV and CTX-M) and point mutations in them, which cause the resistance of microorganisms that cause infectious diseases to penicillins and cephalosporins. For this, it is proposed to use a DNA microchip with specific immobilized oligonucleotides and a multi-primer polymerase chain reaction for the simultaneous amplification of beta-lactamase genes of the indicated types. In the process of PCR, a label - biotin is introduced into the beta-lactamase genes. Biotin is detected on the surface of the microchip using the highly specific interaction of biotin with streptavidin as part of a covalent conjugate with horseradish peroxidase, which is then detected colorimetrically by the accumulation of an insoluble colored product of the enzymatic reaction adsorbed on the surface of the microchip. To quantify the result of hybridization on a chip, it is possible to use high-resolution optical scanners.

Specific oligonucleotides and control oligonucleotides are immobilized simultaneously in the form of a matrix on the surface of the microchip from an insoluble carrier, which can be used glass, porous membrane carriers and other materials. General view of the DNA chip and the location of specific oligonucleotides on it are shown in Fig.1. Each oligonucleotide is applied in the form of three spots located in a horizontal line. The size of the spots is determined by the parameters of the equipment used and the principle of application. Immobilization and hybridization controls are applied to lines restricting the microchip from above and below or to the right and left. Oligonucleotides for detecting one point mutation are arranged in the form of two lines, each of three points, one under the other. The number of lines for determining one mutation increases if there are several variants of gene polymorphism in the study area. As specific oligonucleotides for identifying STX-M type beta-lactamases genes and identifying 238/240 mutation positions in SHV type beta-lactamases, a 1: 1 mixture of two oligonucleotides is used (a mixture of oligonucleotides 30 and 31 for identifying STX beta-lactamases genes -M type, a mixture of oligonucleotides 20 and 21 for the position corresponding to the wild-type enzyme SHV-1, a mixture of oligonucleotides 22 and 23 and a mixture of oligonucleotides 28 and 29 for mutants S_238 / 240_MT_1 and S_238 / 240_MT_6, respectively).

The method of conducting hybridization analysis includes the following stages:

a) the stage of DNA extraction from a clinical sample;

b) the stage of simultaneous amplification of the beta-lactamase genes TEM, SHV and CTX-M types by the method of multiplex polymerase chain reaction with the introduction of the label - biotin;

C) the stage of fragmentation of labeled DNA using DNase;

d) the stage of hybridization of labeled DNA with oligonucleotides immobilized on a DNA microchip;

e) the stage of detection of the biotin label, consisting of incubation of the microchip with the streptavidin-HRP conjugate, followed by colorimetric HR detection.

As a result of the hybridization analysis of DNA samples isolated from clinical samples of pathogens, the following technical result is achieved on a microchip with an immobilized set of oligonucleotides:

1) the specific amplification of full-sized genes of beta-lactamases of various types in one multiplex PCR with the simultaneous introduction of biotin as a label,

2) highly specific simultaneous determination of beta-lactamase genes TEM, SHV and CTX-M types and key mutations in them. The use of biotin as a label allows one to provide higher specificity for the determination of point mutations due to the small size of the molecule and less influence on the conformation of the polynucleotide chain in comparison with fluorescent labels.

The principle of conducting gabridization analysis on a DNA microarray allows you to expand the composition of oligonucleotides and add specific nucleotides to identify other mutations in the TEM, SHV and CTX-M types of beta-lactamases or to identify other types of beta-lactamases while maintaining the method of analysis.

The proposed method is simple and reliable, it does not use expensive reagents and equipment.

List of abbreviations:

BLRS - extended-spectrum beta-lactamases

HRP - horseradish peroxidase

PCR - polymerase chain reaction

Examples confirming the possibility of carrying out the invention

The following examples are intended only to confirm the feasibility of the invention and do not limit the scope of protection of the invention.

Example 1

Identification of the TEM type beta-lactamase gene and determination of point mutations by hybridization analysis on a DNA microarray with colorimetric detection

Oligonucleotides were covalently immobilized on the surface of a nitrocellulose microchip according to the scheme shown in Fig. 1 from solutions with a concentration of 20 μM in saline buffer (160 mm Na ₂ SO ₄ , 130 mm Na ₂ HPO ₄ ) by applying spots in the form of spots with a diameter from 50 to 350 μm using a mini-plotter "Xact Arrayer" (LabNEXT Inc, USA), then incubated for 30 min at 60 ° C. The microchip was washed 5 min in 0.1% TritonX-100, 4 min in a 0.05% HCl solution, 10 min in 100 mM KCl with constant shaking on a horizontal shaker at room temperature. After that, the microchip was washed for 5 min in PBS (0.01 M KH ₂ PO ₄ , 0.15 M NaCl, pH 7.0) and incubated in a 1% BSA solution in the same buffer at 37 ° C for 30 min to block free protein binding sites on the surface of the microchip.

To isolate DNA, the studied enterobacterial samples were seeded to separate colonies on agar plates. The cups were incubated in a thermostat at 37 ° C for 12-15 hours. Then 2-3 identical colonies (1/2 loops) were transferred using a sterile 1 μl loop into a 0.5 ml centrifuge tube with 400 μl of sterile deionized water, suspended and microbial cells were pelleted by centrifugation for 2 min at 7000 g. The supernatant was then removed and the pellet resuspended in 100 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Tubes were incubated in a water bath at 99 ° C for 20 minutes. The tube was shaken and centrifuged at 7000 g for 3 min, then the supernatant was used for PCR.

PCR amplification was carried out in 0.5 ml tubes in a volume of 25 μl. The composition of the reaction mixture included: 1 × Taq DNA polymerase buffer (2.5 mm magnesium acetate, 50 mm KCl, 10 mm Tris-HCl, pH 8.3), 2.5 units Taq DNA polymerase, 100 μM dATP, dGTP, dCTP, 60 μM dTTP, 40 μM dUTP-biotin, 0.4 μM direct primer mixes (5'-ATGAGTATTCAACATTTCCGTGTCG-3 ', 5'-TTATATTCGCCTGTGTATTATCTC-3 -ATGGTTAAAAAATCACTGCGCCAG-3 ', 5'-ATGATGACTCAGAGCATTCGCC-3', 5'-GGTGACAAAGAGAGTGCAACGG-3 ') and reverse primers (5'-CTTAATCAGTGAGGCACCTATCTC-3', 5GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCGTCGTGTGTGTGTGTCGTGTGTGTGTGTCGTCGTGTGTCGTCGTGTCGTC 5'-CCGTGGGTTACGATTTTCGCCG-3 ', 5'-CCCTTCGGCGATGATTCTCGC-3') and 5 μl DNA sample solution. Amplification was carried out in a Mastercycler gradient thermocycler (Eppendorf, Germany) according to the following protocol: denaturation for 2 min at 94 ° С, then 30 cycles of amplification (20 sec denaturation at 94 ° С, 30 sec annealing of primers at 65 ° С, 45 sec. Elongation at 72 ° C), then 8 min final elongation at 72 ° C.

For fragmentation, the amplified beta-lactamase gene was dissolved at a concentration of 30 ng / μl in the reaction buffer (40 mM Tris-HCl, 10 mM MgSO ₄ , 1 mM CaCl ₂ , pH 8.0) and DNase was added at a concentration of 0.2 mU / ng DNA. Fragmentation was carried out at room temperature for 5 minutes. The reaction was stopped by adding 3 mm EDTA and incubating for 10 min in an incubator at 65 ° C.

For hybridization, 500 ng of fragmented labeled DNA was dissolved in 400 μl of hybridization buffer (2 × SSPE (0.8 M NaCl, 50 mM NaH ₂ PO ₄ , 5 mM EDTA), pH 7.7, containing 0.2% sodium dodecyl sulfate). The hybridization mixture was applied to a DNA microarray and incubated for 2 hours at a temperature of 45 ° C. After hybridization, the microchip was washed in three stages: 1) 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate containing 0.1% SDS, 2) 2 × SSC and 3) 0.2 × SSC, each stage 10 min at room temperature. Then, the microarrays were incubated in a solution of streptavidin peroxidase conjugate (1/1000 dilution) in PBST (0.01 M KH ₂ PO ₄ , 0.15 M NaCl, 0.05% Tween20 pH 7.0) containing 1% BSA for 1 hours at room temperature with shaking on a horizontal shaker. After incubation, the microchips were washed (2 times, 5 min in FBS, 1 time - 5 min in PBS (0.01 M KH ₂ PO ₄ , 0.15 M NaCl, pH 7.0)) and placed in a substrate solution containing 9.5 ml of 0.1 M acetate buffer pH 5.5, 0.75 ml of a solution of 3.3 ', 5.5'-tetramethylbenzidine (6.3 mg / ml of dimethyl sulfoxide), 1.5 ml of 5% dextran sodium sulfate and 0.075 ml 0, 2 M hydrogen peroxide, for 10 min at room temperature. After staining, the microchips were washed with distilled water and dried in air. Next, the microchips were scanned on a Perfection V750 Pro optical scanner (Epson) at a resolution of 4800 dpi. The resulting image (in tiff format) was processed quantitatively using the Scan Array Express program (PerkinElmer, version 3.0). The results are presented in figure 2.

Example 2

SHV beta-lactamase gene identification and point mutation detection by hybridization analysis on a DNA microarray with colorimetric detection

The microchip was prepared as described in example 1. Isolation of DNA from the sample, amplification of the gene by PCR, fragmentation of the gene, hybridization on a DNA microarray and detection were performed as described in example 1.

The results are presented in figure 3.

Example 3

STX-M type beta-lactamase gene identification and determination of point mutations by hybridization analysis on a DNA microarray with colorimetric detection

The microchip was prepared as described in example 1. Isolation of DNA from the sample, amplification of the gene by PCR, fragmentation of the gene, hybridization on a DNA microarray and detection were performed as described in example 1.

The results are presented in figure 4.

Example 4

Identification of a mixture of three genes of beta-lactamases TEM, SHV and CTX-M types and determination of point mutations in them by hybridization analysis on a DNA microarray with colorimetric detection

The microchip was prepared as described in example 1. Isolation of DNA from the sample, amplification of the gene by PCR, fragmentation of the gene, hybridization on a DNA microarray and detection were performed as described in example 1.

The results are presented in figure 5.

In the table. 1 and 2 show oligonucleotides and types of primers.

Claims (1)

A method for identifying extended class A beta-lactamases genes, which provides for the selection of oligonucleotide sequences identical to the structure of the gene being determined, their simultaneous immobilization on the surface of microarrays, DNA extraction from the sample, DNA amplification with simultaneous labeling, DNA fragmentation using DNase, labeled DNA hybridization with immobilized oligonucleotides followed by label detection, characterized in that as specific oligonucleotides for gene identification beta-lactamase TEM, SHV and CTX-M types and the detection of point mutations in the following oligonucleotides are used:
room The name of the oligonucleotide 5 '- 3' sequence one ID_TEM NH2-TTTTTTTTTTTTT-CCAGAAACGCTGGTGAAAGT 2 T_104_WT NH2-TTTTTTTTTTTTT-GAATGAGTTGGTTGAGTACTCACCAG 3 T_104_MT NH2-TTTTTTTTTTTTT-GAATGACTTGGTTAAGTACTCACCAG four T_164_WT NH2-TTTTTTTTTTTTT-CGCCTTGATCGTTGGGAACC 5 T_164_MT_1 NH2-TTTTTTTTTTTTT-CGCCTTGATAGTTGGGAACC 6 T_164_MT_2 NH2-TTTTTTTTTTTTT-CGCCTTGATTGTTGGGAACC 7 T_164_MT_3 NH2-TTTTTTTTTTTTT-CGGCTTGATCATTGGGAACCG 8 T_238_WT NH2-TTTTTTTTTTTTT-TСTGGAGGCGGTGAGCGTG
9 T_238_MT_1 NH2-TTTTTTTTTTTTT-TCTGGAGCCAGTGAGCGTG 10 T_238_MT_2 NH2-TTTTTTTTTTTTT-CTGGAGCCGATGAGCGTGG eleven T_240_WT NH2-TTTTTTTTTTTTT-GGAGCCGGTGAGCGTGGGT 12 T_240_MT NH2-TTTTTTTTTTTTT-GGAGCCGGTAAGCGTGGGT 13 ID_SHV NH2-TTTTTTTTTTTTT-GTTGATCCGCTCCGTGCTG fourteen S_35_WT NH2-TTTTTTTTTTTTT-GAGCAAATTAAACTAAGCGAAAGCCAG fifteen S_35_MT NH2-TTTTTTTTTTTTT-GAGCAAATTAAACAAAGCGAAAGCCAG 16 S_179_WT NH2-TTTTTTTTTTTTT-GCGACGCCCACGACACCACTAC 17 S_179_MT_1 NH2-TTTTTTTTTTTTT-GCGACGCCCACAACACCACTAC eighteen S_179_MT_2 NH2-TTTTTTTTTTTTT-CGACGCCCACGGCACCACTACC 19 S_179_MT_3 NH2-TTTTTTTTTTTTT-CGACGCCCACGCCACCACTACC twenty S_238 / 240_WT NH2-TTTTTTTTTTTTT-CCGGAGCTGGTGAACGAGGTGC 21 NH2-TTTTTTTTTTTTT-CCGGAGCTGGTGAGCGAGGTGC 22 S_238 / 240_MT_1 NH2-TTTTTTTTTTTTT-CCGGAGCTAGCAAACGAGGTGC 23 NH2-TTTTTTTTTTTTT-CCGGAGCTAGCAAGCGAGGTGC 24 S_238 / 240_MT_2 NH2-TTTTTTTTTTTTT-CGGAGCTAGCGAGCGAGGTG 25 S_238 / 240_MT_3 NH2-TTTTTTTTTTTTT-CGGAGCTGGCAAACGAGGTGC 26 S_238 / 240_MT_4 NH2-TTTTTTTTTTTTT-CGGAGCTAGCAGGCGAGGTGC 27 S_238 / 240_MT_5 NH2-TTTTTTTTTTTTT-CGGAGCTGCCAAACGAGGTGC 28 S_238 / 240_MT_6 NH2-TTTTTTTTTTTTT-CGGAGCTGCCGAACGAGGTGC 29th NH2-TTTTTTTTTTTTT-CGGAGCTGCCGAGCGAGGTGC thirty ID_STX-M NH2-TTTTTTTTTTTTT-ATATCGCGGTGATCTGGCC
31 NH2-TTTTTTTTTTTTT-ATATCGCGGTTATCTGGCC 32 ID_CTX-M-1 NH2-TTTTTTTTTTTTT-CACCCAGCCTCAACCTAA 33 ID_CTX-M-2 NH2-TTTTTTTTTTTTT-TTACCCAACCGGAGCAGA 34 ID_CTX-M-8 NH2-TTTTTTTTTTTTT-CACCCAGCCAGAGCAGAA 35 ID_CTX-M-9 NH2-TTTTTTTTTTTTT-TACCCAGCCGCAACAGA 36 C1_167_WT NH2-TTTTTTTTTTTTT-UTTTAACGTCUGCTCGGTACGGT 37 C1_167_MT_1 NH2-TTTTTTTTTTTTT-GTTTAACGTCGTCTCGGTACGGT 38 C1_167_MT_2 NH2-TTTTTTTTTTTTT-GTTTAACGTCGACTCGGTACGGT 39 C1_240_WT NH2-TTTTTTTTTTTTT-GGCAGCGGTGACTATGGCAC 40 C1_240_MT NH2-TTTTTTTTTTTTT-GGCAGCGGTGGCTATGGCAC 41 C2_26_WT NH2-TTTTTTTTTTTTT-CAACGCTGCATGCGCAGGCG 42 C2_26_MT NH2-TTTTTTTTTTTTT-CAACGCTGCACGCGCAGGCG 43 C2_230_WT NH2-TTTTTTTTTTTTT-CGAAATCATGGGTAGTGGGCGATA 44 C2_230_MT NH2-TTTTTTTTTTTTT-CGAAATCATGGGGAGTGGGCGATA 45 C2_240_WT NH2-TTTTTTTTTTTTT-CGGCAGCAGAGATTATGGCACCA 46 C2_240_MT NH2-TTTTTTTTTTTTT-CGGCAGCAGAGGTTATGGCACCA 47 C2_253_WT NH2-TTTTTTTTTTTTT-TATCTGGCCGGAAAACCACGC 48 C2_253_MT NH2-TTTTTTTTTTTTT-TATCTGGCCGGGAAACCACGC 49 C2_278_WT NH2-TTTTTTTTTTTTT-CGCAGCCAGAATATCCCGACGG fifty C2_278_MT NH2-TTTTTTTTTTTTT-CGCAGCCAGAACATCCCGACGG 51 C9_121_WT NH2-TTTTTTTTTTTTT-ACGCTGGCAGAGCTGAACGCG 52 C9_121_MT NH2-TTTTTTTTTTTTT-ACGCTGGCAGAACTGAACGCG
53 C9_167_WT NH2-TTTTTTTTTTTTT-TTCAGCGTAGGTTCAGTGC 54 C9_167_MT NH2-TTTTTTTTTTTTT-TTCAGCGTAGATTCAGTGC 55 C9_231_WT NH2-TTTTTTTTTTTTT-TCGTGGACTGCAGGTGATAAGA 56 C9_231_MT NH2-TTTTTTTTTTTTT-TCGTGGACTGTAGGTGATAAGA 57 C9_240_WT NH2-TTTTTTTTTTTTT-GGCAGCGACGACTACGGCACCA 58 C9_240_MT NH2-TTTTTTTTTTTTT-GGCAGCGACGGCTACGGCACCA 59 Sc NH2-TTTTTTTTTTTTT-TCTAGACAGCCACTCATA-Biotin 60 PHC NH2-TTTTTTTTTTTTT-GATTGGACGAGTCAGGAGC 61 NHC NH2-TTTTTTTTTTTTT-TCTAGACAGCCACTCATA
DNA amplification is carried out by the method of multi-primer PCR, and the following are used as primers: direct 5'-ATGAGTATTCAACATTTCCGTGTCG-3 ', 5'-TTATATTCGCCTGTGTATTATCTC-3', 5'-ATGGTTAAAAAATCACTGCGCCAG-3 ', 5', 5AT -GGTGACAAAGAGAGTGCAACGG-3 ', inverse 5'-CTTAATCAGTGAGGCACCTATCTC-3', 5'-GTTAGCGTTGCCAGTGCTCG-3 ', 5'-CCGTCGGTGACGATTTTTAGCCG-3', 5'-CCGTGTGTGTGCCGGCCGTGTGTGCCGGGGGGGG-3 ' Biotin is used as a DNA label, the detection of which is carried out colorimetrically using the horseradish streptavidin-peroxidase conjugate.

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