RU2415937C1 - Dna microchip for identifying extended spectrum class a beta-lactamase genes - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: genes and mutations therein are indentified on a DNA-microchip by hybridisation analysis, wherein a marked DNA is hybridised with immobilised oligonucleotides to assess thereafter mark activity in DNA duplexes on a carrier surface. The 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. ^ EFFECT: new compounds show effective biological properties. ^ 4 dwg, 1 tbl, 4 ex

Description

The invention relates to the field of molecular biology and analytical biotechnology and can be used in clinical microbiological laboratories and epidemiological control services to detect the resistance of Enterobacteriaceae microorganisms to antibiotics of the penicillins and cephalosporins group by the presence of mutations in coding genes.

Antibiotics of the penicillin and cephalosporin groups are currently the most widely used in the treatment of infectious diseases caused by gram-negative microorganisms of the Enterobacteriaceae family. The main factor limiting their clinical effectiveness is antibiotic resistance, i.e. the ability of microorganisms to develop resistance to their action. The main cause of resistance is enzymatic hydrolysis with beta-lactamase enzymes formed in the cell wall [Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev., 2005, 18, pp. 657-686]. 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 the plasmid localization of genes, the distribution of these enzymes among pathogens of human infectious diseases, especially infections, is increasing every year. The most famous and common types of BLRS are the types 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.

A promising method for identifying genes is microarray genotyping methods. DNA microarrays are a substrate of glass, silicon, porous membrane materials, polymer carriers onto which synthetic oligonucleotides or DNA fragments are applied in a matrix. The technology of DNA microarrays for identifying genes and mutations in them is based on the amplification of the required nucleic acid region with simultaneous labeling and subsequent hybridization of the studied nucleotide sequence on the microchip surface with known oligonucleotides or DNA sequences immobilized in a specific order on the microchip [Heller MJ. DNA microarray technology: devices, systems, and applications. Annu Rev Biomed Eng. 2002, 4, 129-53]. The result of hybridization of labeled DNA of the sample with immobilized oligonucleotides is determined by the activity of the label in different parts of the carrier using special devices. 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 has been described to identify STX-M type beta-lactamase genes and individual mutations in them by hybridization analysis [Ensor V.M., Livermore D.M., Hawkey P.M. A novel reverse-line hybridization assay for identifying genotypes of CTX-M-type extended-spectrum P-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, and as a result, only a limited number of mutations can be determined. 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 microarray for the identification of TEM type beta-lactamase genes and mutations in them by hybridization analysis with fluorescence detection [European Patent 1580281 A2, date of publication September 28, 2005 Bulletin 2005/39]. On this glass microchip, oligonucleotides are immobilized that are identical in structure to different regions of the TEM-1 beta-lactamase gene and its mutants; identification is carried out by hybridization analysis with fluorescence detection.

The objective of the invention is the development of a DNA microarray with immobilized specific oligonucleotides for the highly specific simultaneous detection of the main types of beta-lactamase class A genes (TEM, SHV and CTX-M) and key point mutations in them, responsible for the development of resistance of microorganisms that cause infectious diseases to antibiotics of penicillins and cephalosporins.

To solve this problem, a molecular design of the structure of oligonucleotides was performed to identify beta-lactamase genes TEM, SHV, and CTX-M types and to determine key mutations in them. To identify the type of gene, a sequence was used that is complementary to the conserved region of genes of this type and does not have homology with genes of other types. To identify the CTX-M type beta-lactamase subcluster, a sequence was used that is complementary to a gene region highly homologous to the genes of one subcluster and with a low degree of homology with genes belonging to other subclusters.

To determine the point mutation, a set of four oligonucleotides with a sequence of bases corresponding to the structure of the beta-lactamase gene in the studied area and differing from each other with a nucleotide in the central position corresponding to the point mutation, which was used as one of the four nucleotides A, G, C, was initially tested. or T. Upon hybridization of the studied DNA with a set of oligonucleotides, the most stable duplex is formed with a fully complementary oligonucleotide and, accordingly, in this case, a higher analytical signal is detected. When duplexes are formed as a result of hybridization with the other three oligonucleotides or only some of them, the observed analytical signal indicates the level of non-specific hybridization. The molecular design consisted of optimizing the length of oligonucleotides and introducing artificial substitutions in their sequence to ensure high specificity of gene identification and determination of mutations while using them in a hybridization reaction carried out under selected conditions at a fixed temperature. The high specificity of the determination of individual mutations in the genes of beta-lactamases TEM, SHV and CTX-M types by the method of hybridization analysis is shown in figure 1. The use of biotin as a DNA label followed by colorimetric detection provided the highest specificity for determining mutations.

In the future, to determine one mutation, a set of two or, in some cases, a larger number of oligonucleotides is used. The sequence of the first oligonucleotide is complementary to the sequence of the gene of the "wild" type, the second or others - the sequence of one or more mutants. To control the processes of immobilization and hybridization on a DNA microarray, control oligonucleotides are used (immobilization control, positive hybridization control and negative hybridization control). The sequence of specific and control oligonucleotides are shown in table 1.

Specific oligonucleotides and control oligonucleotides are immobilized simultaneously in the form of a matrix on the surface of microchips 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.2. 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 limiting 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 genes, a 1: 1 mixture of two oligonucleotides is used (a mixture of oligonucleotides 30 and 31 for identifying STX-M type beta-lactamases , 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 determination of beta-lactamase genes and point mutations in them is carried out by the method of hybridization analysis on a DNA microarray:

1. Isolation of DNA from a clinical sample;

2. Amplification of DNA by PCR with the simultaneous introduction of biotin tags;

3. Fragmentation of labeled DNA using DNase;

4. Hybridization of labeled DNA with oligonucleotides immobilized on a DNA microchip;

5. Detection of label activity on the surface of the microchip;

6. Obtaining a microchip image on a scanner and analyzing the results.

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

highly specific simultaneous determination of beta-lactamase genes TEM, SHV and CTX-M types and key mutations in them.

The composition of oligonucleotides on a DNA microarray can be expanded by adding new 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.

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

Comparison of the specificity of determining point mutations in beta-lactamase genes by hybridization analysis on microarrays with fluorescence and colorimetric detection.

Oligonucleotides from solutions with a concentration of 20 μM in salt buffer (160 mM Na ₂ SO ₄ , 130 mM Na ₂ HPO ₄ ) are immobilized on the surface of a microchip of glass with epoxy groups or a porous membrane carrier by applying spots to the microchip in the form of spots with diameters from 50 to 350 μm using a mini-plotter "Xact Arrayer" (LabNEXT Inc, USA). Each oligonucleotide is applied in the form of three spots located in a horizontal line. After application, glass microchips are incubated in a thermostat for 30 min at 60 ° C, washed for 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. To block free epoxy groups, microarrays are incubated in a blocking solution (25% ethylene glycol solution containing 0.05% HCl) at 50 ° C for 15 min with constant stirring, then washed in deionized water for 1 min and dried.

After application, the microarrays from the membrane carrier are incubated in a thermostat for 30 minutes at 60 ° C, then washed in deionized water for 1 minute and dried.

For hybridization, 500 ng of fragmented biotin-labeled or fluorescent Su3 tag 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% dodecyl sulfate sodium). 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, fluorescence detection microarrays were scanned on a ScanArray scanner (Perkin Elmer) with a resolution of 5 μm. Microchips with biotin as a label were incubated in a solution of streptavidin peroxidase conjugate (1/1000 dilution) in PBS (0.01 M KN ₂ PO ₄ , 0.15 M NaCl, 0.05% Tween20 pH 7.0) containing 1% BSA , for 1 hour at room temperature with shaking on a horizontal shaker. After incubation, the microchips were washed (2 times, 5 min in FBST, 1 time - 5 min in PBS (0.01 M KN 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 obtained images of hybridization results (in tiff format) were processed quantitatively using the Scan Array Express program (PerkinElmer, version 3.0). The results are presented in figure 1.

Example 2

Immobilization of specific oligonucleotides on the surface of a glass microchip.

Oligonucleotides from solutions with a concentration of 20 μM in salt buffer (160 mM Na ₂ SO ₄ , 130 mM Na ₂ HPO ₄ ) are immobilized on the surface of a microchip made of glass with epoxy groups or a porous membrane nostel. μm using a mini-plotter "Xact Arrayer" (LabNEXT Inc, USA) according to the scheme shown in figure 2. Each oligonucleotide is applied in the form of three spots located in a horizontal line. Oligonucleotides for detecting one point mutation are arranged in the form of two lines, each of three points, under each other. As specific oligonucleotides for identifying STX-M type beta-lactamases genes and identifying 238/240 mutation positions in SHV type genes, a 1: 1 mixture of two oligonucleotides is used (a mixture of oligonucleotides 30 and 31 for identifying STX-M type beta-lactamases , 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).

After application, glass microchips are incubated in a thermostat for 30 min at 60 ° C, washed for 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. To block free epoxy groups, microarrays are incubated in a blocking solution (25% ethylene glycol solution containing 0.05% HCl) at 50 ° C for 15 min with constant stirring, then washed in deionized water for 1 min and dried.

After application, the microarrays from the membrane carrier are incubated in a thermostat for 30 minutes at 60 ° C, then washed in deionized water for 1 minute and dried.

Example 3

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

To isolate DNA, the studied samples of enterobacteria were scattered 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) μl DNA sample solution. Amplification was carried out in a Mastercycler gradient amplifier (Eppendorf, Germany) according to the following protocol: denaturation for 2 min at 94 ° C, then 30 cycles of amplification (20 sec denaturation at 94 ° C, 30 sec annealing of primers at 55 ° C, 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 PBS (0.01 M KN ₃ 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 3.

Example 4

Identification of a mixture of TEM, SHV and CTX-M beta-lactamase genes and determination of point mutations by hybridization analysis on a DNA microarray with colorimetric detection.

A microchip was prepared as described in example 2. DNA was extracted from the sample as described in example 3. Amplification by PCR was carried out in 0.5 ml tubes in a volume of 25 μl. 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. Fragmentation of the gene, hybridization on a DNA microarray and detection were performed as described in example 3. The results are presented in figure 4.

Claims (1)

DNA microarray to identify the genes of beta-lactamase extended-spectrum class based on glass or a porous membrane carrier having immobilized on the surface of oligonucleotides comprising oligonucleotides NH2 TTTTTTTTTTTTTTSTAGASAGSSASTSATA-biotin to control immobilization, NH2-TTTTTTTTTTTTTGATTGGACGAGTCAGGAGC for positive hybridization control, NH2-TTTTTTTTTTTTTTCTAGACAGCCACTCATA for a negative control of hybridization, characterized in that as specific oligonucleotides for the identification of beta-lactamase TEM, SHV and CT genes XM types and detection of point mutations in them 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-GAATGACTTGGTTGAGTACTCACCAG 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-CGCCTTGATCATTGGGAACCG 8 T_238_WT NH2-TTTTTTTTTTTTT-TСTGGAGCCGGTGAGCGTG 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_CTX-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-GTTTAACGTCGGCTCGGTACGGT 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-GGGCAGCAGAGATTATGGCACCA 46 C2_240_MT NH2-TTTTTTTTTTTTT-CGGCAGCAGAGGTTATGGCACGA 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

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