RU2455364C2 - Method of identifying mycobacteria by polymerase chain reaction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves the PCR-amplification of the 16S rRNA fragment with the use of specific original oligonucleotide primers SEQ ID No: 1 and SEQ ID NO: 2. It is followed by the PCR biotinylation of the primer SEQ ID No: 1 which enables detecting the nucleotide sequences of the 16S rRNA by simple one-chain solid-phase sequencing with the use of specific original sequencing primers SEQ ID No: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The mycobacteria are precisely identified by comparing certain sequences with common sequences of various types of mycobacteria in the database.

EFFECT: invention enables identifying mycobacterioses in the samples of pathological material.

4 dwg, 1 tbl, 2 ex

Description

A method for identifying mycobacteria using a polymerase chain reaction relates to epizootology, microbiology, biotechnology and medicine. The proposed identification method is intended for the identification of mycobacteria in clinical and pathological material by polymerase chain reaction using specific oligonucleotides corresponding to regions of pRNA genes. Automated and simple DNA sequencing methods are provided.

Standard methods for accurately identifying mycobacterial species in accordance with phenotypic characteristics include complex and lengthy procedures. Methods based on lipid analysis, such as liquid, thin-layer, gas-liquid chromatography, are cumbersome, expensive and are used only in several clinical laboratories in Europe. Not only Mycobacterium tuberculosis and Mycobacterium bovis, but also other mycobacteria are becoming increasingly important as opportunistic pathogens that can cause a number of serious diseases. The most striking example is the spread of Mycobacterium avium and the recently described Mycobacterium genavense in patients with AIDS.

There is a great need for quick identification of all types of mycobacteria.

A known method for detecting the DNA of mycobacterium tuberculosis complex [RU 2163638 C1, 7 C12Q 1/68, 12/06/1999] and similar methods [RU 94017389 A1, 6 C12Q 1/68, 05/10/1994], [RU 97100857 A1, 6 C12Q 1 / 68, 01/24/1997], which do not allow for the species identification of all types of mycobacteria.

Identification of nucleotide sequences is a quick and widely used method, but it covers only a narrow range of mycobacterial species; in addition, there are problems associated with specificity and sensitivity. Molecular genetic methods for typing microorganisms are known, such as restriction fragment length polymorphism analysis and dot blot hybridization, however, the method for determining the 16S pRNA sequence is universal [Rogall, T., Flohr, T., and Böttger, E.S. (1990) Differentiation of mycobacterium species by direct sequensing of amplified DNA. J. Gen. Microbiol. 136, 1915-1920].

Genes for 16S and 23S rRNAs are particularly suitable for identifying microorganisms because (except for viruses) rRNA genes are found in all organisms. Ribosomal nucleic acids are regarded as phylogenetically significant molecules.

The separation of bacteria into sections, classes, families, genera, and species is reflected in the pRNA sequence. RRNA sequences or “molecular imprints” are the basis for the identification of microorganisms. “Molecular imprints” take into account not only the primary, but also the secondary structure of ribosomal genes.

In contrast to phenotypic and biochemical characteristics, the 16S pRNA sequence of an individual is a stable characteristic that is specific for microorganisms at the species level. Moreover, for the identification of the species, hypervariable regions are especially important, since only low variability in them is observed within the species. Analytical nucleic acid sequencing involves nucleic acid isolation, polymerase chain reaction (PCR) - established multiplication of a 16S pRNA gene fragment, sequence determination and data analysis. Sequencing of 16S pRNA allows one to correctly identify isolates, that is, to identify mycobacteria in an already established taxon, as well as quickly identify still unrecognized species [Seung-Jung Kee, Shin-Mook Kim, Soo-Hyun Kim, Myung-Geun Shin, Jong -Hee Shin, Soon-Pal Suh and Dong-Wook Ryang. (2009) Multiplex PCR Assay for Identification of Mycobacterial Species Isolated from Liquid Cultures. Chonnam Medical Journal. Vol.45, No.1, p.19-26].

After isolation of the nucleic acid by simple mechanical destruction of the bacteria, an amplification of a 16 kD fragment of 1 KD is performed using primers SEQ ID No: 2 and SEQ ID No: 1 (table 1 and figure 1). The high specificity of the primer SEQ ID No: 2 allows accurate identification of mycobacteria in contaminated cultures. Biotinylation of PCR primer SEQ ID No: 1 allows the use of simple single-stranded solid phase sequencing. For a laboratory that has an automated DNA sequencer, the protocol for the Applied Biosystems 373A sequencer is attached (see subheading 3.3). Accurate identification is possible by comparing either specific sequences with known sequences or specific sequences with baseline data. Sequencing methods are highly reliable methods that can be easily implemented in any large clinical laboratory.

Full sequencing does not require large time expenditures (no more than 36 hours, from the moment of receiving mycobacterial culture to the final identification). With modern equipment, amplification and sequencing can be carried out in one day.

Annex 1

1. Materials for sample preparation, PCR amplification and sequencing

1.1. Sample preparation and PCR amplification:

1.1.1. Large tissue disintegrator;

1.1.2. Amplifier;

1.1.3. Agarose gel electrophoresis equipment:

- Acid-treated glass beads with a diameter of 100 mm;

- TE buffer: 10 mM TriS-HCl, pH 8.3 1 mM EDTA;

10X PCR buffer (GeneAmp): 100 mM Tris-HCl, pH 8.3; 500 mM KCl, 15 mM MgCl ₂ , 0.01% gelatin;

- Ampli Taq DNA polymerase;

- dNTP mixture: 1.25 mM of each deoxynucleoside triphosphate;

- Oligonucleotides (see table. 1);

- Double autoclaved water;

- Mineral oil;

- Orange G buffer: 0.25% orange G, 30% glycerol in TE;

- molecular weight marker;

- TBE buffer: 89 mM Trisborate, 89 mM boric acid, 8 mM EDTA (ethylenediaminetetraacetic acid).

1.2. Automated DNA sequencing:

1.2.1. Applied Biosystems 373A DNA sequencer;

1.2.2. Amplifier Perkin Elmer GeneAmp 9600 or other amplifier;

1.2.3. Vacuum centrifuge;

1.2.4. Prism staining of the terminal sequencing cycle with a ready-made reaction complex (Applied Biosystems);

1.2.5. 0.2 ml GeneAmp PCR tubes;

1.2.6. QIA quick PCR purification kit;

1.2.7. 3A sodium acetate, pH 5.2;

1.2.8. Ethanol;

1.2.9. Formamide, 50 mM EDTA, pH 8.0 (5: 1).

1.3. Simple DNA sequencing:

1.3.1. Sequencing equipment;

1.3.2. Dynal MPC-E magnetic separator;

1.3.3. Dynabeads M-280 Steptovidin;

1.3.4. Sequencing kit, for example, Seguenase version 2.0 DNA sequencing kit;

1.3.5. Sequencing Buffer: 200 mM Tris, pH 7.5; 100 mM MgCl ₂ ;

1.3.6. Labeled mixture: 0.2 mM dATF; 0.2 mM dTTF; 0.2 mM dHTF;

1.3.7. dTCF, 3000 Ci / mmol, 10 mCi / ml;

1.3.8. Stop buffer: 95% formamide (deionized), 20 mM EDTA, bromphenol blue until saturated color forms;

1.3.9. Acrylamide;

1.3.10. Urea;

1.3.11. Darkroom;

1.3.12. X-ray installation.

2. Methods for sample preparation, PCR amplification and sequencing

2.1. Sample preparation:

2.1.1. For amplification by PCR, 0.5 ml of a liquid culture (12B WASTES, Becton-Dickinson) is taken from 10 different cultures of mycobacteria with a growth index of 50.0, or a colony from a medium (e.g. Löwenstein-Jensen) dissolved in 0.5 ml TE, or microscopically positive condensed liquid with solid egg medium Löwenstein-Jensen;

2.1.2. Place the sample in 1.5 ml microcentrifuge tubes with a screw cap. Incubated for 10 min at 80 ° C in order to inactivate mycobacteria;

2.1.3. Centrifuge the tubes at maximum speed (approximately 17,000 g) in a microcentrifuge for 10 minutes. Remove the supernatant;

2.1.4. Add 100 μl TE and loop glass beads. Tubes are placed in a large tissue disintegrator and subjected to processing at maximum speed for 2 minutes;

2.1.5. Centrifuge the sample at maximum speed in a microcentrifuge for 5 minutes;

2.1.6. Pour the top layer into a new microcentrifuge tube and use 5 μl for PCR.

2.2. PCR amplification:

2.2.1. Prepare the reaction mixture for PCR. 10 reactions are carried out and used in this case:

- 50.0 μl 10X GeneAmp. PCR buffer;

- 80.0 μl of a mixture of dNTP;

- 10.0 μl of biotinylated primer SEQ ID No: 1 (8 pmol / ml);

- 10 μl of primer SEQ ID No: 2 (20 pmol / ml);

- 297.5 μl of sterile water;

- 2.5 μl of Taq polymerase;

2.2.2. 45.0 μl of the reaction mixture are placed in 0.5 ml microcentrifuge tubes and a drop of mineral oil is added to each;

2.2.3. The tubes are placed in an amplifier at a temperature of 72 ° C;

2.2.4. Add 5.0 μl of sample to the prepared mixture;

2.2.5. Amplification is carried out according to the following protocol:

- 94 ° C, 1 min (denaturation);

- 64 ° C, 2 min (annealing) 40 cycles;

- 72 ° C, 2 min (synthesis);

- 72 ° C, 5 min (final stage);

2.2.6. Take 5.0 μl (aliquot) of the mixture and add 5.0 μl of orange G buffer;

2.2.7. Prepared samples are applied to 0.8% agarose gel in TBE buffer and electrophoresis is performed. The orange marker moved about 6-10 cm. The amplification product was visualized in UV light. The resulting PCR products were analyzed by automated direct or simple DNA sequencing.

2.3. Automated DNA sequencing.

The following procedure is used for Taq cyclic sequencing on Applied Biosystems 373A DNA sequencer:

2.3.1. Purify the double-stranded PCR product from primers and nucleotides using the QIAquick PCR purification kit as indicated by the manufacturer. Then, a column of purified PCR product is washed from QIAquick in 50.0 μl of sterile water;

2.3.2. The components for Taq cyclic sequencing reaction are mixed in a 0.2 ml GeneAmp tube:

- 5.0 μl of purified PCR product (corresponding to 100-150 ng of DNA);

- 8.0 μl of the final finished reaction compound (ABI PRISM dye of the final sequencing cycle);

- 2.0 μl of sequencing oligonucleotide primers SEQ ID No: 3 (5 pmol / μl);

- 5.0 μl of water (demineralized);

2.3.3. Place the tube in a Perkin Elmer GeneAmp 9600 amplifier and amplify according to the scheme:

- 25 cycles: 96 ° C, 10 min;

- 60 ° C, 4 min;

- after the last cycle, the sample is kept at 4 ° C;

2.3.4. The following components are added to the prepared 0.5 ml microcentrifuge tube for each reaction:

- 2.0 μl of 3 M sodium acetate, pH 5.2;

- 50.0 μl of 95% ethanol;

2.3.5. Transfer all 20.0 ml of the contents of the reaction tube to tubes containing ethanol solution. Mix and place on ice for 10 min;

2.3.6. Centrifuge the tube at maximum speed in a microcentrifuge for 15 minutes;

2.3.7. Carefully select the ethanol solution with a pipette;

2.3.8. Rinse the bead, add 250.0 μl of 70% ethanol. Centrifuge the tube in a microcentrifuge with a maximum acceleration of 3 minutes. Carefully select the entire alcohol solution;

2.3.9. In a vacuum centrifuge, the bead is dried for 3-5 minutes;

2.3.10. Re-place the bead in 4.0 μl of formamide / 50 mM EDTA (5: 1);

2.3.11. Incubated for 2 min at 90 ° C, followed by placement on magnesium and sequencing;

2.3.12. Gel sequencing is carried out according to the instructions using Applied Biosystems;

2.3.13. Perform fluorescence studies based on Applied Biosystems software.

2.4. DNA sequencing:

2.4.1. To prepare a single-stranded DNA sample (using biotinylated primer SEQ ID No: 1), 20.0 μl of the PCR reaction product (see Subheading 3.2) and 10.0 μl of Dynabeads M280 streptavidin are mixed using a Dynal MPC-E magnetic separator (according to the manufacturer's instructions) ) Re-place the beads with single-stranded linked DNA in 10.0 ml of water;

2.4.2. Sequencing components are mixed in a 1.5 ml reaction tube:

2-5 μl Dynabead single-stranded DNA solution (bead suspension from step 1);

- 1.0 μl of sequencing primers SEQ ID No: 4 or SEQ ID No: 5 (3 pmol / ml) 2.0 μl of sequencing buffer;

- 1.0 μl NP 40 (5%);

- adjusted with water to a volume of 10.0 μl;

2.4.3. Annealing for 20 min at 37 ° C in a water bath;

2.4.4. The following is added to the annealed primer compound:

- 1.0 ml 0.1 M DTT;

- 2.0 ml marker mixture;

- 2.0 ml sequencer (dissolved in TE 1: 8);

- 0.5 ml of α-dCTP (3000 Ci / mmol) dissolved in TE 1: 6 (you can prepare the mixture for several reactions and add it to 5.5 ml);

2.4.5. Incubated for 5 min at room temperature;

2.4.6. 3.0 μl (aliquot) of the sequencing reaction was transferred to four 1.5 ml tubes to which 2.5 ml of the deoxy / dideoxy final compound was added. Each compound contains 80 mM dATP, 80 mM dCTP, 80 mM dGTP, 80 mM dTTP and 8 mM corresponding dideoxynucleotide triphosphate;

2.4.7. The final compound tubes are incubated for 5 min at 37 ° C, then 4.0 μl of buffer is added. You must make sure that the buffer reaches the bottom of the tubes with the suspension of the magnetic bead;

2.4.8. Tubes are incubated for 5 min at 75 ° C and freshly synthesized DNA fibers are washed from beads;

2.4.9. Use a Dynal MPC-E magnetic separator to bind the beads, and load 3.0 μl of the reaction mixture (without beads) into a 6% urea polyacrylamide gel — sequencing;

2.4.10. Sequencing

2.4.11. The analysis results are recorded using x-ray film.

Analyze the data of the studies by comparing a certain sequence with the alignment of mycobacterial sequences, including the hypervariable region A (transmitting E. coli 16S pRNA position from 129 to 266, see Figs. 1 and 2) and the hypervariable region B (E. coli 16S pRNA position from 430 to 497, see FIG. 3). In this case, the research results coincide with the table. Of the 10 cultures of pathogenic and atypical mycobacteria, all were classified with 100% coincidence.

Appendix 2

For a positive WASTES 13A environment, the blood culture is poured into mattresses, subcultures are prepared to dissolve the blood, or the guanidine isothiocyanate / phenolic method is used to prepare samples, because the remaining blood components make PCR difficult.

Cross-contamination must be controlled using negative control. Prevent contamination with pre-amplified PCR products using precautions such as physical separation of the various steps, various pipette sets, aerosol-free tips and wearing separate lab coats and gloves in each laboratory.

The biotinylation of primer SEQ ID No: 1 and its restriction to a size of 8 pmol is necessary only with simple sequencing.

All amplifiers according to technical data are different and the amplification conditions change depending on this. Monitoring the correct implementation of amplification is the serial dilution of a positive control, for example, aqueous mycobacterial suspension (Mc.Farland, standard 0.5). Aliquots of 1: 100 dilutions can be stored at -20 ° C. Each time a new dissolved positive control is used.

To identify culture isolates using simple sequencing, the use of primer SEQ ID No: 4 is sufficient for sequencing region A. Sometimes, additional primer SEQ ID No: 5 is used for region B.

If the ribosomal sequence of the isolate is not included in the alignment (Figure 1-3), then search for the current database of sequences, for example, GenBank. In the GenBank database, a stream of 16S pRNA sequences is available via email. World Wide Web and on CD-ROM (PC / Gene, IntelliGenetics Inc., Mountain View, CA). The "Ribosomal Database Design" provides a variety of software and analysis of user-submitted sequences via email, FTP, and the World Wide Web.

Mixed mycobacterial infections can be determined by the presence of several sequences, i.e. the simultaneous presence of bands in different lines of the sequencing gel during simple sequencing.

Close species of M. kansasii and M. gastri show an identical 16S pRNA sequence; these two taxa can be distinguished by determining the sequence of 16S-23S pRNA region. M. ulcerans and M. marinum show an almost identical 16S pRNA gene sequence, but the hypervariable regions A and B allow these taxa to be differentiated. In contrast, M. gordonae shows species-specific ribosomal sequence variation within region A. This discovery of genomic species within a species is comparable to the analysis of a restriction enzyme enhanced by the hsp-65 gene fragment. M. avium has one nucleotide sequence variable within the 16S pRNA gene at position 135 (position corresponding to E. coli).

Claims (1)

A method for identifying mycobacteria using a polymerase chain reaction, characterized in that PCR amplification of the 16S rRNA fragment is carried out using specific original oligonucleotide primers SEQ ID No: 1 and SEQ ID No: 2, followed by biotinylation of the PCR primer SEQ ID No: 1, which allows to determine 16S pRNA nucleotide sequences by simple single-stranded solid-phase sequencing using specific original sequencing primers SEQ ID No: 3, SEQ ID No: 4 and SEQ ID No: 5 and carry out accurate identification of mycobacteria ery comparing certain sequences with known sequences of different species of mycobacteria in the database.

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