Method of simultaneous amplification and fluorescent marking of several genome segments of mycobacteria of tuberculosis complex

FIELD: biotechnology.

SUBSTANCE: invention provides method of simultaneous (multiplex) amplification and fluorescent marking of DNA of several segments of the genome of mycobacteria of tuberculosis complex (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum and M. microti). Then the hybridisation or electrophoretic analysis of the sequences of these segments is carried out. The multiplex PCR is carried out in a single reaction volume with use of specific and adapter primers, the fluorescent substrate embedded into the growing DNA chain during PCR, and genomic DNA as a matrix. The multiplex PCR comprises two consecutive amplification profiles, different in annealing temperatures of specific and adapter primers by not less than 10°C. The invention enables to carry out the analysis of sequences of these genes to identify mutations associated with resistance to anti-tuberculosis preparations.

EFFECT: method according to this invention reduces the number of stages of amplifications to obtain single stranded fluorescent-labelled PCR-products, eliminates the transfer of a DNA-matrix from one reaction volume to another, which increases the stability of the procedure to contamination with PCR-products and reduces significantly the time and complexity of the analysis.

4 dwg, 1 tbl, 1 ex

 

The invention relates to molecular biology, Microbiology and medicine and provides a method for simultaneous amplification and fluorescent labeling of DNA several segments of the genome of Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum and M. microti for subsequent hybridization or electrophoretic analysis of sequences of data segments.

The level of technology

Currently, the world health organization (who) identifies the causative agents of tuberculosis and multidrug-resistant TB (MDR TB MDR TB), extensively drug-resistant (XDR-TB, XDR TB) and infectious agents, with a total (complete) resistance to anti-TB drugs (extensively drug resistant tuberculosis TB, XXDR TB). MDR TB is caused by mycobacteria that are resistant at least to the two most effective first-line drugs - isoniazid and rifampicin. XDR-TB is a form of tuberculosis, the pathogen which is resistant to isoniazid and rifampicin, one of the drugs fluoroquinolone, and at least one of three injectable anti-TB drugs second-line treatment: amikacin, kanamycin or capreomycin (WHO, Global tuberculosis control : WHO report 2012., in WHO/HTM/TB/2012.62012, WHO: Geneva. p. 1-98).

Molecular analysis of the genome of Mycobacterium tuberculosis has been successfully implemented in the practice of the TB control service of the Russian �federazii and abroad. Developed diagnostic tests for the detection of DNA of Mycobacterium tuberculosis with simultaneous identification of mutations associated with drug resistance of the pathogen to various anti-TB drugs (Pai M, Minion J, Sohn H, Zwerling A, Perkins MD. Novel and improved technologies for tuberculosis diagnosis: progress and challenges. Clin Chest Med 30(4), 701-716, viii (2009)).

Methods for the identification of point mutations, based on PCR with detection in real time, such as recommended by the world health organization automated Xpert MTB/RIF system (Cepheid, USA), define only a limited number of mutations in rpoB gene that lead to rifampicin resistance and cannot be used for the detection of MDR strains and, especially, XDR (List of publications on this system is available on the website of the who on the following link: Published evidence and commentary on the Xpert MTB/RIF assay).

Hybridization technology in the format of "one patient - one tube - one of the detecting medium (nitrocellulose membrane or a biological microchip)" identify a significantly larger number of mutations in various segments of the mycobacterial genome, thereby identify strains with MDR and partly with XDR. The test procedure must include one or two-stage multiplex PCR using mycobacterial DNA in Kutch�as the matrix, followed by hybridization of the PCR products on the detecting medium.

The test-system "GenoType MTBDRplus" (Hain LifeSciences, Germany) based on one-step multiplex PCR followed by hybridization of PCR products with the DNA probes on the membranes (strips), which gives the opportunity to detect DNA of Mycobacterium in sputum and determine the four most common mutations in the rpoB gene (rifampicin resistance) and the genes katG and inhA (isoniazid resistance). The main disadvantage of this test is its low analytical sensitivity (10,000 cells in 1 ml of sample), which allows to apply the method for rapid detection of MDR-strains in the sputum with a positive microscopy result (Bq+) or in cultures highlighted in the liquid and/or dense nutrient media (Paolo Miotto, Federica Piana, Daniela Maria Cirillo, Giovanni Battista Migliori. Genotype MTBDRplus: a further step toward rapid identification of drug-resistant Mycobacterium tuberculosis. https://www.researchgate.net/journal/1098-660X_Journal_of_clinical_microbiology 2008; 46(1):393-4).

Reagent kits series "TB-Biochip" (Institute of molecular biology. V. A. Engelgardt, Russia) for identification of Mycobacterium tuberculosis and determination of its sensitivity to rifampicin and isoniazid ("TB-Biochip-1", Patent RF 2376387; Gryadunov D., V. Mikhailovich, S. Lapa, N. Roudinskii, M. Donnikov, S. Pan kov, O. Markova, A. Kuz'min, Chernousova L., O. Skotnikova, A. Moroz, A. Zasedatelev and A. Mirzabekov. Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis. Clinical microbiology and infection 2005. Vol 11:p.531-539), to identify sosbud�the determinant of TB and determination of its sensitivity to the fluoroquinolones ("TB-BIOCHIP-2", O. V. Antonova, D. A. Gryadunov, Lapa S. A., A. V. Kuzmin, E. E. Larionova, T. G. Smirnova, E. Yu., Nosov, O. I. Skotnikov, L. N. Chernousova, A. M. Moroz, A. S. Zasedatelev, V. M. Mikhailovich. The identification of mutations in the genome of Mycobacterium tuberculosis, resulting in resistance to fluoroquinolones, the method of hybridization on biological microchips. Bulletin of experimental biology and medicine 2008. No. 1. p. 115-120) use a two-step multiplex PCR followed by hybridization on the biological microchip containing immobilized oligonucleotides that are specific to different mutations. Methods have a high analytical sensitivity (500 cells in 1 ml of sample), however, the two-stage multiplex PCR with unavoidable products of the first stage in the test tube containing the reaction mixture of the second stage PCR, electrophoretic control of each stage substantially complicate the analysis procedure, requires specialized equipment and highly qualified personnel, increase the time and increase the risk of contamination of the reaction mixtures of PCR products, leading to false-positive results.

Thus, in this area there is an urgent need to develop a method of amplification of several segments of the mycobacterial genome, which would favorably known in the tech� solutions ease of analysis (the number of stages of amplification - no more than one, no phase electrophoresis), high analytical sensitivity and resistance to contamination of PCR products.

Disclosure of the invention

Advantages of the method of simultaneous amplification and fluorescent labeling of multiple segments of the genome of Mycobacterium tuberculosis complex.

Method of simultaneous amplification and fluorescent labeling of multiple segments of the genome of Mycobacterium tuberculosis complex favorably known in the art methods of conducting multiplex amplification in a single reaction volume (the tube or hole tablet) with simultaneous inclusion of fluorescent labels during PCR and obtaining at the output of the single-stranded fluorescently-labeled PCR products that can be further analyzed using electrophoresis and/or by hybridization to the oligonucleotide microarray (microarray). The method does not require several successive stages of PCR in different reaction volumes (tubes or the wells of the microplate), in which the first phase involves the amplification of fragments that are specific to segments of the mycobacterial genome, and at subsequent stages - accumulation of single-stranded products with their simultaneous fluorescent labeling. The proposed method reduces Chi�lo stages of amplification to obtain single-stranded flourescence-labeled PCR products, eliminates the DNA transfer matrix from one reaction space to another, which increases the resistance to contamination of PCR products, and reduces the complexity of the analysis. The method is designed, primarily, as a stage of analysis of sequences of genes antibioticresistance of Mycobacterium tuberculosis complex, for the purpose of establishment of mutations associated with resistance to various anti-TB drugs.

In its first aspect the present invention provides a method for simultaneous amplification and fluorescent labeling of multiple segments of the genome of Mycobacterium tuberculosis complex, carried out in a single closed reaction volume, containing at least two pairs of specific primers and a pair of adapter primers, and includes:

(a) amplification profile that uses:

- at least two pairs of specific primers, the sequence of which is partially complementary sequences of at least two independent segments of the mycobacterial genome;

- genomic DNA of mycobacteria as template for PCR.

components of the reaction mixture, providing the time and fluorescent labeling of PCR products, which includes specific segments of the mycobacterial genome and are flanked on the 5'ends of the �posledovatelnostei, complementary to the adapter sequences of the primers are:

b) the amplification profile that uses:

- a pair of adapter primers, the sequence of which is complementary to the sequences of fragments that are located at the 5'ends of the PCR products of step (a);

RT - PCR products obtained in stage (a) serving as a matrix for PCR;

components of the reaction mixture, providing the time and fluorescent labeling of PCR products, which includes specific segments of the mycobacterial genome.

In one of its embodiments the method is characterized in that all components of PCR mixture for both profiles of amplification, including the genomic DNA of mycobacteria-specific and adaptor primers are located in a single reaction volume.

In the following embodiment of the method is characterized in that both the amplification profile account for one-stage PCR, and the profiles themselves are performed sequentially in a single reaction volume.

In the following embodiment of the method is characterized in that the annealing temperature of the specific primers of the first amplification profile exceeds the annealing temperature of the adapter primers of the second profile is not less than 10 ° C.

In the following embodiment of the method is characterized in that the sequence of each of specific primers consists of two f�of Ahmetov, the sequence of the fragment comprising the 5'-end primer, a complementary sequence of one of the adapter primers, and the sequence of the fragment comprising the 3'-end-specific primer complementary to a sequence segment of the mycobacterial genome.

In the following embodiment of the method is characterized in that the concentration of each of specific primers in the reaction mixture is less than the concentration of adapter primers at least 50 times.

In the following embodiment of the method is characterized in that the concentration of forward and reverse adapter primers in the reaction mixture may vary 5 or more times, for the purpose of obtaining predominantly single-stranded PCR products.

In one of the embodiments the method is characterized in that the fluorescent labeling of PCR products during the PCR is carried out using the fluorescent dye conjugate and deoxynucleotides added to the reaction mixture.

Finally in one of the embodiments the method is characterized in that the obtained PCR products can be used for further hybridization or electrophoretic analysis.

Other aspects of the present invention will become apparent from the accompanying figures, detailed description and claims.

A summary list of figures

For b�more clear understanding of the essence of the claimed invention and also to demonstrate its distinctive features and benefits the following is a detailed description of the invention with reference to the figures of drawings, on which:

Fig. 1. represents the scheme of the multiplex PCR in the claimed method.

Fig. 2. is electrophoresis picture of PCR products obtained when performing multiplex PCR, stated in this way, and when using the reference method.

Fig. 3. is an example of hybridization of PCR products on a specialized oligonucleotide microchip obtained when performing multiplex PCR, stated in this way, using as template the DNA of M. tuberculosis wild-type.

Fig. 4. is an example of hybridization of PCR products on a specialized oligonucleotide microchip obtained when performing multiplex PCR, stated in this way, using as template the DNA of M. tuberculosis containing mutations resulting in amino acid substitutions Ser531>Leu in rpoB gene and Ser315>Thr in the gene katG.

The implementation of the invention

The object of the present invention is to provide a method for simultaneous amplification and fluorescent labeling of multiple segments of the genome of Mycobacterium tuberculosis complex.

To ensure efficient amplification with simultaneous introduction of FL�orescent label all fragments of a variant of the multiplex PCR using specially designed primers (see Fig.1). The sequence of each added to the reaction mixture of specific primer (Fis, Risi - room amplificative segment) consists of two parts - a 3'-specific, i.e. complementary sequence of a fragment of the genome of Mycobacterium tuberculosis complex, and a 5'-universal (adapter), distinguished as forward and reverse primers. In addition to these primers, and containing specific, and the adapter sequence in the reaction mixture add two primers (Fa,Ra), the sequence of which is complementary to the adapter sequences of part-specific primers. The concentration of specific primers is less than the concentration of adapter primers at least 50 times.

Forward and reverse adapter primers present in the reaction mixture in a proportion of not less than 1:5, aimed at finding a predominantly single-stranded fragments. This condition is necessary, for example, to sequence single-stranded fragments which were complementary to the sequences of oligonucleotides immobilized oligonucleotide in the cells of the microchip.

Sequence-specific and adaptor primers chosen so that their calculated melting temperature Tmsand Tmadiffered �e less than 10°C, ie (Tms- Tma) ≥10°C. In this case, the amplification profile may include two-stage PCR in a single reaction volume (the microtube or the wells of the microplate), different annealing temperatures of the primers is not less than 10°C. at the first stage baked and completed only the specific primers having a high melting point and dwindling by the end of the first stage, and by lowering the annealing temperature by 10°C in the second stage PCR start working adapter primers.

Thus, during PCR in a single reaction volume at the first profile of amplification by hybridization and elongation-specific primers using genomic DNA as template occurs time between double-stranded PCR products containing at the ends of the sequence specific to the adapter primers, and then, changing profile of amplification, the obtained PCR products serve as a matrix for producing single-stranded fragments using adapter primers with a lower annealing temperature. This amplification scheme is illustrated in Fig. 1.

Fluorescent labeling of the studied fragments of the mycobacterial genome is carried out by adding to the PCR mixture with a fluorescent substrate, such as conjugate deoxyuridylate and dye indodicarbocyanine range, with lengths�wave excitation, equal (640±5) nm, and the wavelength of the fluorescence equal to (665±5) nm. During PCR, the substrate is embedded Taq-DNA polymerase into a growing DNA chain, providing at the output of fluorescently-labeled PCR products analysed further by electrophoresis or hybridization at a specialized oligonucleotide microchip.

Hereinafter the invention will be illustrated by example, which is intended to provide a better understanding of the essence of the claimed invention, but should not be construed as limiting the invention.

Example

Simultaneous amplification of a fragment of insertion element IS6110, fragments of genes rpoB gene, katG, inhA, ahpC M. tuberculosis. The results of electrophoresis and hybridization to a specialized oligonucleotide microchip for identification of mutations in the genome of Mycobacterium tuberculosis complex associated with resistance to rifampicin and isoniazid. Evaluation of analytical sensitivity.

Using the algorithm presented above is designed primers for simultaneous amplification of a fragment of insertion element IS6110, fragments of genes rpoB gene, katG, inhA, ahpC. Sequence-specific and adaptor primers involved in a multiplex PCR for amplification of the above segments of M. tuberculosis, the concentration of primers in the reaction mixture privedennyu Table 1.

Table 1. Sequence-specific and adaptor primers, their length and the concentration used for the simultaneous amplification of a fragment of insertion element IS6110, fragments of genes rpoB gene, katG, inhA, ahpC.

PrimerSequence (5' to 3')Length, NCConcentration, nm
rpoB gene-Fs*ctctgcgagcataatg-agtgagacgggtgcacgtcgcggacct3910
rpoB gene-Rsggctgtacgctgtc-gttgatcaacatccggccggtggtcgc3410
katG-Fsctctgcgagcataatg-tggaagagctcgtatggcaccggaa4310

katG-Rsggctgtacgctgtc-cggtgtattgccaagcgccagcag4110
inhA-Fsctctgcgagcataatg-ccggaaatcgcagccacgttacgc4010
inhA-Rsggctgtacgctgtc-ggtaaccaggactgaagggatacgaa 3710
ahpC-Fsctctgcgagcataatg-atggtgtgatatatcacctttgcctgacagc4410
ahpC-Rsggctgtacgctgtc-tgactctcctcatcatcaaagcggaca4210
Is-Fsctctgcgagcataatg-aggatggggtcatgtcaggtggttcatcga4610
Is-Rsggctgtacgctgtc-gcgaagaaagccgacgcggtctttaaaatc3610
uni-Factctgcgagcataatg16500
uni-Raggctgtacgctgtc142500

*The designation. F - direct primer, R - reverse primer, index 's' indicates specific primer, the index 'a' - adapter

Primers were chosen so that the length of the resulting PCR products fragments of the studied segments of the mycobacterial genome had the following values: IS6110 - 309 nucleotides (NC), rpoB gene - 212 NC, katG -166 NC, inhA - 133 NC, ahpC - 106 NC.

The reaction mixture (3 μl) contained 3 μl of buffer for HS-Taq polymerase (CJSC "Evrogen, Russia), 3 ál of 2 mm mixture of deoxynucleotides (CJSC "Silex", Russia), 3 µl of a mixture of primers specified in Table 1 concentrations of 8 µm fluorescent substrate IMD515-dUTP (eimb RAS, Russia), 1 µl HS-Taq-DNA-polymerase (CJSC Evrogen, Russia) and 3 µl of the sample genomic DNA of M. tuberculosis.

Used the following PCR profile: denaturation at 95°C, the first profile of amplification of 40 cycles (95°C - 30 s, 64°C 30 s, 72°C - 30 s) , followed by a second amplification profile was 40 cycles (95°C - 30 s, 50°C - 30 s, 72°C - 30 s) and 72°C - 5 min.

As a method of comparison used methods of analysis of the genome of Mycobacterium tuberculosis complex from a Set of reagents for the detection of Mycobacterium tuberculosis and to determine their drug resistance to rifampicin and isoniazid by hybridization on the biological microchip "TB-Biochip 1 according to THE 9398-001-02699501-2010 (Registration certificate of Federal service on surveillance in healthcare and social development of the Russian Federation No. SDF 2011/10088) described (D. Gryadunov, V. Mikhailovich, S. Lapa, N. Roudinskii, M. Donnikov, S. Pan kov, O. Markova, A. Kuz'min, Chernousova L., O. Skotnikova, A. Moroz, A. Zasedatelev and A. Mirzabekov. Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis. Clinical microbiology and infection 2005. Vol 11:p.531-539) and patented (Mirzabekov, A. D., Mihajlovic, V. M., Sobolev, A. Y. gryadunov D. A., Lapa S. A., Zasedatelev A. S. Method for simultaneous detection of bacteria tubercu�a serious complex and identification of mutations in the DNA of mycobacteria, resulting in resistance of microorganisms to rifampicin and isoniazid, on biological microchips, a set of primers, a biochip, and a set of oligonucleotide probes used in the method. Patent RF 2376387. Priority from 26.12.2005. Published 20.12.2009) earlier. The technique is based on two-stage PCR to simultaneous amplification of fragments of genes rpoB gene, katG, inhA, ahpC and insertion element IS6110, followed by hybridization on a specialized biological microchip. The products of both PCR stages are subject to mandatory electrophoretic control. The PCR products obtained in the first stage PCR, are the matrix for the second stage PCR (1 μl reaction mixture containing PCR products of the first stage is transferred to a separate tube for the second stage PCR). The length of PCR products, fragments corresponding to the genes rpoB gene, katG, inhA, ahpC and insertion element IS6110 obtained in the first stage PCR, similar to those specified above.

The results of amplification of mycobacterial DNA using comparison method and the inventive multiplex PCR procedure in the form of electrophoretic pattern in 4% agarose gel shown in Fig. 2. Tracks:

1 - molecular marker of the lengths of the fragments;

2 - the result of amplification of the genes rpoB gene, katG, inhA, ahpC and insertion element IS6110 using the "TB-Biochip-1 and DNA-M. tuberculosis wild type as template;

3 - the result of amplification of the genes rpoB gene, katG, inhA, ahpC and insertion element IS6110 using the "TB-Biochip-1 and M. tuberculosis DNA containing mutations resulting in amino acid substitutions Ser531>Leu in rpoB gene and Ser315>Thr in the gene katG;

4 - the result of amplification of the genes rpoB gene, katG, inhA, ahpC and insertion element IS6110 obtained when performing multiplex PCR, stated in this way, when using DNA of M. tuberculosis wild type as template;

5 - the result of amplification of the genes rpoB gene, katG, inhA, ahpC and insertion element IS6110 using the "TB-Biochip-1 and M. tuberculosis DNA containing mutations resulting in amino acid substitutions Ser531>Leu in rpoB gene and Ser315>Thr in the gene katG.

As evidenced by the results presented in Fig. 2, a variant of the multiplex PCR, claimed in the present method allows the simultaneous amplification of five segments of the mycobacterial genome.

For evaluating the effectiveness of amplification and fluorescent labeling of PCR products obtained by using a variation of multiplex PCR, claimed in the present method, hybridisable on a specialized oligonucleotide microchip for identification of mutations in the genes rpoB gene, katG, inhA, ahpC genome of M. tuberculosis associated with resistance to rifampicin and isoniazid, and incoming � the composition of a set of reagents "TB-Biochip-1". Hybridization was performed as described previously (D. Gryadunov, V. Mikhailovich, S. Lapa, N. Roudinskii, M. Donnikov, S. Pan kov, O. Markova, A. Kuz'min, Chernousova L., O. Skotnikova, A. Moroz, A. Zasedatelev and A. Mirzabekov. Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis. Clinical microbiology and infection 2005. Vol 11:p.531-539).

The results of hybridization of PCR products obtained by using multiplex PCR, claimed in the present method and DNA of M. tuberculosis wild-type shown in Fig. 3.

In accordance with the previously described algorithm for interpreting the results of hybridization (Mirzabekov, A. D., Mihajlovic, V. M., Sobolev, A. Y. gryadunov D. A., Lapa S. A., Zasedatelev A. S. Patent RF 2376387. Priority from 26.12.2005. Published 20.12.2009) cells with oligonucleotides complementary DNA of the wild type, have a greater intensity of fluorescent signal than the other cells within each group. Thus, the DNA of the studied object forms a perfect hybridization duplexes with oligonucleotides complementary to the DNA sequence of wild-type (sensitive to rifampicin and isoniazid strain of Mycobacterium tuberculosis). Accessory DNA of this strain to the Mycobacterium tuberculosis complex is established by the presence of fluorescent signal in cells with immobilized oligonucleotide on IS6110 (F8, I8). Perfect hybridization duplexes recorded in all �the group of cells containing probes specific to sequences of the genes rpoB gene, katG, inhA, ahpC, element IS6110, which means uniform amplification and fluorescent labeling using a multiplex PCR, claimed in this way.

The results of hybridization of PCR products obtained by using multiplex PCR, claimed in the present method and DNA of M. tuberculosis containing mutations resulting in amino acid substitutions Ser531>Leu in rpoB gene and Ser315>Thr in the gene katG shown in Fig.4.

In each of the groups of elements of the biochip, the intensity of the fluorescence signal in the cell with an oligonucleotide complementary DNA of the wild type, higher signal intensity of the remaining cells. The exception is the group of elements of D3 - D7 (1) and E1,E2, F1 - F7 (2) (Fig. 4).

Maximum intensity of fluorescence within the group D3-D7 has an element of D4. Therefore, DNA of the studied object has a point nucleotide substitution A>T in the position 2431 gene rpoB gene (Genbank Acc. No. L27989) that leads to the substitution of amino acids Ser to Leu at position No. 531 and leads to the emergence of resistance of the studied strain to rifampicin. Maximum intensity of fluorescence within the group E1,E2, F1-F7 has the element E2. Thus, to determine the presence in the studied DNA base substitutions (G on a C at position 1013 katG gene (Genbank Acc. No. U06262) that leads to the substitution of the amino acid�you Ser 315 for Thr at position No. 315 and causes the resistance of the studied strain to isoniazid. Accessory DNA of this strain to the Mycobacterium tuberculosis complex is established by the presence of fluorescent signal in cells with immobilized oligonucleotide on IS6110 (F8, I8). Perfect hybridization duplexes recorded in all groups of cells containing probes specific to sequences of the genes rpoB gene, katG, inhA, ahpC, element IS6110, which means uniform amplification and fluorescent labeling using a multiplex PCR, claimed in this way.

The analytical sensitivity of the developed method was evaluated by serial tenfold dilutions of DNA of the strain of M. tuberculosis H37 RV and analysis, according to the procedure described above. The proposed method allows to reproducibly detect at least 300 genome-equivalents of DNA of Mycobacterium tuberculosis complex (which corresponds to the concentration of 100 genome equivalents in 1 ml sample) and to identify in it the presence of mutations responsible for resistance to anti-TB drugs. In this case the analytical sensitivity of the reference method set "TB-Biochip-1" is 500 genome equivalents.

Thus, the proposed in the present method variant multiplex PCR allows for effective simultaneous amplification and fluorescent labeling of several (in �esteem example five segments of the mycobacterial genome. The inventive method has a high analytical sensitivity comparable to the reference method, it does not require the holding of a two-stage amplification in different reaction volumes, with the obligatory electrophoretic control each stage of the PCR and the migration of amplification products of the first stage in the test tube containing the reaction mixture for the second stage PCR.

The method of this invention compares favorably with existing analogues reduced to one stage of amplification to obtain single-stranded flourescence-labeled PCR products, excludes the DNA transfer matrix from one reaction space to another, which increases the resistance to contamination of PCR products, and reduces the complexity of analysis.

Although the preferred embodiment of the present invention and their advantages have been described in detail above, the person skilled in the art can make various modifications, additions, or, conversely, something lower, though still within the scope of this invention, which is defined below by the claims.

Method of simultaneous amplification and fluorescent labeling of multiple segments of the genome of Mycobacterium tuberculosis complex, carried out in a single closed reaction volume�e (the microtube or the hole of the tablet), containing at least two pairs of specific primers and a pair of adapter primers and a fluorescent dye conjugate and discountlevitraonline providing fluorescent labeling, and includes:
(a) amplification Stage, which occurs time and fluorescent labeling of PCR products, which includes specific segments of the mycobacterial genome and are flanked on the 5'ends of the sequences, complementary to the adapter sequences of the primers and uses:
- at least two pairs of specific primers, the sequence of each of which is composed of two fragments, the sequence of the fragment comprising the 5'-end primer, a complementary sequence of one of the adapter primers, and the sequence of the fragment comprising the 3'-end-specific primer complementary to a sequence segment of the mycobacterial genome, wherein the concentration of each of specific primers in the reaction mixture is less than the concentration of adapter primers at least 50 times.
- genomic DNA of mycobacteria as a matrix for PCR;
components of the reaction mixture, providing the time and fluorescent labeling of PCR products, which includes specific segments of the mycobacterial genome and Flanker�data on 5'ends of the sequences, complementary to the adapter sequences of the primers;
b) a Stage of amplification, which occurs time and fluorescent labeling of PCR products, which includes specific segments of the mycobacterial genome, and uses:
- a pair of adapter primers, the sequence of which is complementary to the sequences of fragments that are located at the 5'-ends of PCR products obtained in stage (a), the difference in annealing temperatures of adapter-specific primer is at least 10°C, and the concentrations of forward and reverse adapter primers in the reaction mixture may vary 5 or more times, for the purpose of obtaining predominantly single-stranded PCR products;
RT - PCR products obtained in stage (a) serving as a matrix for PCR;
components of the reaction mixture, providing the time and fluorescent labeling of PCR products, which includes specific segments of the mycobacterial genome.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: invention represents pCFP10-DBD recombinant plasmide consisting of an artificial bacterial operon of a chimeric protein including a promoter region of early promoter of T5 bacteriophage, a chimeric protein gene consisting of a sequence of protein antigene CFP10 from Mycobacterium tuberculosis, which is merged with a sequence of a dextrane-binding domain (DBD) of dextrane sucrase Leuconostoc citreum KM20, and a transcription terminator, a bacterial operon of beta-lactamase and a bacterial initiation section of replication of ColE1 type. Besides, strain Escherichia coli - a producer of CFP10-DBD chimeric protein is presented. The invention describes a method of immobilisation, concentration and cleaning of the obtained protein on cellulose. The invention describes an immunogenic composition containing CFP10-DBD recombinant protein, which is aimed at induction of immunity against tuberculous infection.

EFFECT: invention enlarges the range of devices used for treatment of tuberculosis.

4 cl, 6 dwg, 1 tbl, 4 ex

FIELD: process engineering.

SUBSTANCE: set of invention relates to biology, particularly, to automated purification of biological specimens at extraction of target matters involving the magnetic field application. Magnetic field application element and heater are integrated for up and down movement. Automatic device comprises unit 100 displacing vertically and horizontally to accommodate pipettes 141, 142 for suction and discharge of fluid. Heater 810 heats a particular unit of holes of multihole tray 420, 420' while element 700 for magnetic field application is arranged on support plate 400 including magnet seat 710. Element 700 accommodates magnet 711 at bottom side of said unit of said tray Proposed method exploits above described device.

EFFECT: higher process efficiency.

26 cl, 48 dwg

FIELD: biotechnology.

SUBSTANCE: bionanoconjugate comprises a nano-sized superparamagnetic particle of cobalt ferrite spinel CoxFe3-xO4, where 0.6≤x≤0.98, obtained by mechanochemical synthesis. To isolate the nucleic acid containing oligo- or poly-A/dA sequence the synthetic single stranded oligonucleotide 5'-dGndTm is used, where n=5-30, m=10-35, one portion of which, consisting of guanine nucleotides in the presence of phosphate anions in the solution is specifically associated with the surface of the nanoparticle and the other - consisting of thymine nucleotides is able to enter into hybridisation with oligo- or poly-A/dA nucleotide sequences. For isolation from the solution in the presence of phosphate anions of specific hetero-nucleotide sequence additionally create a molecule of oligonucleotide-adapter containing the sequence of oligo-dAx at the 3'-end, hybridised with thymine nucleotides of a complex 5'-dGndTm, where n=5-30, m=10-35, and a portion at the 5'-end complementary to a specific hetero-nucleotide sequence in the solution.

EFFECT: invention enables to detect effectively and to isolate the single-stranded nucleic acids.

3 cl, 8 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, in particular to methods of obtaining enzyme preparations. Invention deals with thermally stable two-domain laccase of bacterium Streptomyces griseoflavus Ac-993 with alkaline optimum of activity, DNA sequence, coding said enzyme, and method of obtaining enzyme, including cloning enzyme gene in cells of bacterium Escherichia coli, production of enzyme in cells of Escherichia coli, introduction of copper ions in medium for cultivation of recombinant strain in order to form active enzyme, obtaining enzyme preparation by methods of metal chelate chromatography and gel filtration.

EFFECT: invention makes it possible to extend assortment of enzymes such as laccase.

3 cl, 3 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to gene engineering, biochemistry, biotechnology and immunology, and represents recombinant plasmid pESAT6-CFP10-DBD, recombinant strain Escherichia coli M15 [pREP4, pESAT6-CFP10-DBD], a method for preparing, immobilising, concentrating and purifying recombinant protein ESAT6-CFP10-DBD on dextran, recombinant protein ESAT6-CFP10-DBD with molecular weight 26.4 kDa, an immunogenic composition containing protein ESAT6-CFP10-DBD immobilised on dextran, specifically activating T-lymphocytes synthesising IFN-gamma with antigen stimulation of mycobacteria.

EFFECT: invention enables ensuring the high level of protein expression, which induces the immune response to mycobacterial antigen effectively.

5 cl, 2 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, in particular to molecular biology, biotechnology, genetic engineering. The invention represents optimised DNA sequences, coding a light and heavy chain of a monoclonal antibody, intended for the synthesis of the antibody in recombinant CHO-S cells, and a method of obtaining the said antibody in the said cells. The performed work resulted in the elaboration of several conditions making it possible to obtain the monoclonal antibody with high productivity.

EFFECT: invention makes it possible to maximise the cell productivity and optimise post-translational modifications of the antibody, in particular glycosylation of heavy chains of the antibody.

3 cl, 2 dwg, 6 tbl

FIELD: chemistry.

SUBSTANCE: claimed invention relates to biotechnology and represents molecular conjugates, capable of binding with nucleic acids (DNA or RNA) for their delivery into cells of mammals, expressing transferring receptors. The said molecular conjugates consist of a polycationic sequence, represented by a modified signal of nuclear localisation of the virus SV40 T-antigen, and a ligand. One of two sequences HAIYPRH or THRPPMWSPVWP is used as ligands of cell receptors. Complexes of the molecular conjugate and nucleic acid are used for obtaining medications for genetic therapy or diagnostics of various diseases. To obtain the said complexes solutions of nucleic acid and the molecular conjugate are poured together with a ratio of charges in the reaction medium from 1:0.63 and lower and used for complex formation of solutions with ionic power less than 300 mM.

EFFECT: claimed invention makes it possible to increase the efficiency of delivering genetic constructions into cells.

12 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to molecular biology and genetic engineering. What is presented is a RNAi molecule for suppressing the thymidilate synthase expression by the action of RNAi, containing a double-stranded RNA domain consisting of a sense chain consisting of a nucleotide sequence presented by SEQ ID NO: 1 hybridised with an anti-sense chain hybridized in the demanding conditions with the sense chain.

EFFECT: molecule can substantially potentiate the antineoplastic action of 5-FU-antineoplastic agent for which reason it can be used in medicine as a part of the antineoplastic therapy.

15 cl, 2 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a combined recombinant protein of the formula: S-L-R, including SR10, SR13, SR15, SdR10, SdR13 or SdR15, which specifically recognises melanoma cells, where S - streptavidin monomer, L - linker having amino-acid sequence Ser-Arg-Asp-Asp-Asp-Asp-Lys containing a restriction site with enteropeptidase and marked as "d", or amino-acid sequence Ser-Arg-Ala-Gly-Ala,R - melanoma-addressing oligopeptide representing R10 having amino-acid sequence Asp-Gly-Ala-Arg-Tyr-Cys-Arg-Gly-Asp-Cys-Phe-Asp-Gly, or R13 having amino-acid sequence Leu-Ser-Gly-Cys-Arg-Gly-Asp-Cys-Phe-Glu-Glu, or R15 having amino-acid sequence Asp-Gly-Phe-Pro-Gly-Cys-Arg-Gly-Asp-Cys-Ser-Gln-Glu. This invention also describes recombinant plasmid DNAs pSR and pSdR for expression of the specified combined proteins, bacterial strains Escherichia coli MG1655/pSR and MG1655/pSdR, producers of the specified combined proteins and a producing method of melanoma-addressing oligopeptide R from combined recombinant proteins SdR10, SdR13 or SdR15.

EFFECT: invention allows producing combined proteins that provide selective and effective binding to receptors on the surface of melanoma cells and can be used in diagnostics and therapy of cancer of a human being.

9 cl, 7 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to the field of biotechnology and is directed to nucleic acid molecules which encode a protein exhibiting properties of a biosensor for detection of hydrogen peroxide in living cells having fluorescence in the red region of the spectrum. The nucleic acid molecules are derived by method of genetic engineering. Protein for detection of hydrogen peroxide has the amino acid sequence shown in SEQ ID NO:2. The host cell and the expression cassette are also provided, comprising the said nucleic acid molecules.

EFFECT: use of the inventions enables to carry out microscopy in thick layers of tissues, and provides the possibility of joint microscopy of several fluorescent structures.

4 cl 7 dwg

FIELD: molecular biology.

SUBSTANCE: the suggested innovation deals with the fact that nucleic acids should be isolated directly out of the sample without pipetting stage but with the help of interconnected reservoirs being prepared beforehand. The above-mentioned vessels should be applied either separately or being interconnected according to standard microtitrating format. The sample should be mixed with a lyzing buffer and nucleic acids are bound with matrix in closed system including, at least, two interconnected reservoirs. Forced movement of sample's mixture and buffer back and forth from one reservoir into another one for several times through narrow passage provides their thorough intermixing. The method provides quick and safe isolation of nucleic acids.

EFFECT: higher efficiency.

44 cl, 4 dwg, 1 ex

FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.

SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.

EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.

7 cl, 12 dwg, 4 tbl, 16 ex

FIELD: genetic engineering, medicine.

SUBSTANCE: invention relates to T-cell receptor sequence being detected in patients with extended sclerosis and is useful in diagnosis and therapy. Oligonucleotide including sequence which represents or is derived from 5'-CTAGGGCGGGCGGGACTCACCTAC-3' or nucleotide sequence being fully complementary thereto. Oligonucleotide together with nuclear acid including nearly 15-30 oligonucleotides, which doesn't comprise oligonucleotide sequence and presents in region from Vβ to Jβ of Vβ13.1 gene in T-cell Vβ13.1-subgroup, wherein oligonucleotide and nuclear acid sequences don't present in the same chain of pair sequences of Vβ13.1 gene, is used in Vβ13.1 gene part amplification. In method for detection of LGRAGLTY motive, which is present in T-cell receptors of T-cell Vβ13.1-subgroup, oligonucleotide is used in combination with labeling particle. Once LGRAGLTY motive is detected, development monitoring and treatment are carried out by removing of LGRAGLTY motive-containing peptide.

EFFECT: simplified methods for detection of LGRAGLTY motive in T-cell receptors and treatment of patients with extended sclerosis.

21 cl, 7 dwg, 3 tbl, 3 ex

FIELD: biotechnology, in particular virulent genes and proteins.

SUBSTANCE: peptide with Neisseria meningitidis antigen activity and polynucleotide encoding the same are used in preparation of drug for prevention or treatment of diseases and conditions associated with Neisseria or gram-positive infections. Antibodies is obtained by using disclosed peptide. Vaccines for prevention or treatment of diseases and conditions associated with Neisseria meningitides contain attenuated mutant strain of Neisseria meningitides having gene mutation, insertion or deletion which disturbs expression of certain nucleotide sequence.

EFFECT: method for prevention or treatment of Neisseria infection with increased effectiveness.

13 cl, 1 tbl

FIELD: genetic engineering, agriculture.

SUBSTANCE: invention relates to a method for preparing transgenic plants. Method involves selection of the strain of the required plant, preparing mother plants and carrying out the multiplication of the vegetable material for preparing leaf explants. Then method involves making a vector for transfer of genetic material expressing the end protein that enhances resistance of plants against phytopathogens followed by the agrobacterial transformation of explants with agrobacterium Agrobacterium tumifaciens and the following selection of transgenic tissue and multiplication of transformants. At the transformation stage method involves stage-by-stage co-cultivation of explants with steps amount taken in the range from 2 to 5 and wherein leaf disks with the ratio of cut length to the area disk is in the range 0.1-0.2 mm/mm2 are used as explants. Invention provides preparing transgenic plants showing the enhanced resistance against phytopathogens and also to enhance frequency of the transient expression, frequency in formation of transgenic tissues, frequency in regeneration of transgenic sprouts, part of direct transformants and to reduce frequency of somaclonal alterations.

EFFECT: improved preparing method, valuable properties of transgenic plants.

5 cl, 9 dwg, 12 tbl, 16 ex

FIELD: biotechnology, biochemistry, amino acids.

SUBSTANCE: invention describes a polynucleotide showing activity of glucose-6-phosphate isomerase and comprising polynucleotide sequence taken among the group including: a) polynucleotide encoding polypeptide that comprises amino acid sequence identical at least by 90% with amino acid sequence represented in SEQ ID NO:2; b) polynucleotide that is complementary with polynucleotides given in sub-paragraph a). Also, invention describes a method for enhancing the metabolism intensity in pentose phosphate cycle by attenuation of pgi gene and a method for preparing L-amino acids. Invention provides preparing L-amino acids with the high effectiveness degree.

EFFECT: improved preparing method, valuable properties of polynucleotide.

16 cl, 7 dwg, 3 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention describes recombinant host-cells comprising a chimerical gene involving the heterologous promoter sequence associated functionally with nucleic acid that encodes at least one polypeptide implicated in biosynthesis of epotilons, or polypeptide expressed by recombinant way and implicated in biosynthesis of epotilons, and Bac clones also. Using the invention provides to realize a method for preparing epotilons with high degree of effectiveness.

EFFECT: valuable properties of host-cell.

9 cl, 6 tbl, 15 ex

FIELD: biotechnology, microbiology, biochemistry, molecular biology.

SUBSTANCE: for preparing l-lysine method involves carrying out fermentation with using corynebacterium strain producing L-lysine wherein polynucleotide comprising the polynucleotide sequence encoding the component H of phosphotransferase system is enhanced. Prepared L-lysine is isolated. The claimed invention provides enhancing the effectiveness of synthesis of L-lysine by corynebacteria.

EFFECT: valuable properties of strain, enhanced effectiveness of amino acid synthesis.

16 cl, 3 dwg, 1 tbl, 5 ex

FIELD: biotechnology, biochemistry.

SUBSTANCE: invention represents enzyme glycosyl hydrolase (variants) produced by hyphamycetes, in particular, by Chrysosporium lucknowense and nucleic acid construction for expression of glycosyl hydrolase also. Invention provides preparing glycosyl hydrolase by culturing fungus Chrysosporium under optimal conditions providing expression and isolation of enzyme with high degree of effectiveness.

EFFECT: valuable biological and biochemical properties of sequences.

19 cl, 2 dwg, 4 tbl, 1 ex

FIELD: molecular biology, agriculture.

SUBSTANCE: invention relates to a method for determination of plants sex at seedling stage used in selection of hop. Sex of hybrid hop plants is determined by method of molecular marking wherein two combinations of oligonucleotide primers are used for carrying out polymerase chain reaction that provide amplification of DNA fragments specific for male plants. Invention enhances effectiveness of selection and allows decreasing consumptions of labor, agents, squares and time.

EFFECT: improved marking method.

3 dwg, 1 tbl

Up!