RU2486251C2 - Method of identification and differentiation of procariotic organisms - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of identification and differentiation of procariotic microorganisms provides amplification of sequence between the gene replicas of transfer riziform-Glu with use of genus-specific primers. The said sequence is a hin-region. The resulting amplification products are compared with similar characteristics of known hin-regions by the number, length and nucleotide sequence.

EFFECT: use of the invention enables to carry out the identification in the sample of both one and several different procariotic organisms simultaneously.

54 dwg, 4 tbl, 2 ex

Description

The invention relates to the field of biotechnology and relates to a method for identifying a single microorganism or simultaneous identification of several different microorganisms in a sample by the nucleotide sequence of the hin region (from Latin Hin - unique, inimitable) located between copies of the same tRNA-Gly genes.

The main molecular biological methods used for differentiation and identification of organisms can be divided into three categories: methods based on restriction, amplification (PCR), and a combination of the latter.

The first group includes the RFLP technique (Polymorphism of the Lengths of Restriction Fragments), which consists in fragmenting the native DNA of the organism under study using restriction enzymes and subsequent separation of the fragments obtained using Pulse Gel Electrophoresis (PFGE) / Schwartz D.C., Cantor C.R. 1984. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 37 (1): 67-75 /. Possessing the original principle of separation of DNA fragments in a gel, nevertheless, this method is very laborious at the stage of production and restriction of whole unfragmented DNA.

The second group includes such techniques as:

AR-PCR (random PCR performed without knowledge of the nucleotide sequence of the body, based on the genomic distribution of random repeats) / Williams J.G.-K., Kubelik A.R., Livak K.J., Rafalski J.A., Tingey S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers // Nucleic Acids Res. 18: 6531-6535 /;

- Ger-PCR (the technique is a complex of primers for sequences that are often repeated in the genomes of enterobacteria, called Box, Rep, Eric) / Versalotic J., Koeuth T., Lupski J.R. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes // Nucleic Acids Res. 19: 6823-6831 /;

- microsatellite PCR (a technique based on the genomic distribution of microsatellite sequences of type (GC) ₄ , (GTG) ₅ , etc.) / Epplen JT, Ammer H., Epplen C. 1991. Oligonucleotide fingerprinting using simple repeat motifs: a convenient, ubiquitously applicable method to detect hypervariability for multiple purposes // In: DNA fingerprinting: approaches and applications. Eds .: G. Burke, G. Dolf, AJ Jeffreys, and R. Wolff, Birkhauser, Basel. P.50-69; Toonen R. 1997. Microsatellites for Ecologists: Non-Radioactive Isolation and Amplification Protocols for microsatellite markers. Version 1. University of California; Ellegren H. 2004. MICROSATELLITES: SIMPLE SEQUENCES WITH COMPLEX EVOLUTION // Nature Reviews Genetics 5, No. 6,435-445 /;

- tRNA-PCR (tDNA-ILP) (a technique based on the genomic distribution of conserved regions of tRNA genes) / Welsh J., McClelland M. 1991. Genomic fingerprints produced by PCR with consensus tRNA gene primers // Nucleic Acids Res. 19: 861-866 /;

- IS-PCR (a technique based on the genomic distribution of sequences of mobile elements found in the genome of the studied organism) / Mahillon J., Chandler M. 1998. Insertion sequences // Microbiol. Mol. Biol. Rev. 62: 725-774; George M. L. C., Bustamam M., Cruz W. T., Leach J. E., Nelson R. J. 1997. Movement of Xanthomonas oryzae pv. oryzae in southeast Asia detected using PCR-based DNA fingerprinting // Phytopathology. 87: 302-309 /;

- PCR analysis with primers for target genes (such genes may be housekeeping genes (gyrB, rpoD, dnaK, fyuA, atpD, recA, ginll, rpoB, etc.) / Berg T., Tesoriero L., Hailstones DL 2005. A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas. // Letters in Appl. Microbiology. 42 (6): 624-630; Parkinson N., Aritua V., Heeney J., Cowie C., Bew JD, Stead D. 2007. Phylogenetic analysis of Xanthomonas species by comparison of partial gyrase B gene sequences // Int. J. of Syst. And Evol. Microbiology. 57: 2881-2887; Maiden MC, Bygraves JA, Feil E. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms // Proc. Natl. Acad. Sci. USA 95: 3140-3145); Vinuesa P., Rojas-Jiménez K., Contreras-Moreira B., Mahna SK, Nandan Prasad B., My H., Babu Selvaraju S., Thierfelder H., Wemer D. 2008. Multilocus sequence analysis for assessment of the biogeography and evolutionary genetics of four Bradyrhizobium species that nodulate soybeans on the asiatic continent // Applied and Environmental Microbiology. 74 (22): 6987-6996 /, ribosomal cluster (16S pPHK, intergenic region located between the 16S pPHK and 23S pPHK genes, 23S pPHK) / Kolbert S.P., Persing D.H. 1999. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens // Curr.Opin. Microbiol. 2: 299-305 /, as well as symbiotic genes (sym genes, nod genes) / Franssen H.J., Nap J.P., Bisseling T. 1992. Nodulins in root nodule development // Biological Nitrogen Fixation. P.598-624; Suominen L., Roos C., Lortet G., Paulin L., Lindström K. 2001. Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer // Mol. Biol. Evol. 18 (6): 907-916 /, genes for pathogenicity (hrp) / Berg T., Tesoriero L., Hailstones D.L. 2005. A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas. // Letters in Appl. Microbiology. 42 (6): 624-630; Leite R.P. (Jr), Minsavage G.V., Bonas U., Stall R.E. 1994. Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria II // Appl. Environ. Microbiol. 60: 1068-1077; Zaccardelli M., Campanile F., Spasiano A., Merighi M. 2007. Detection and identification of the crucifer pathogen, Xanthomonas campestris pv. campestris, by PCR amplification of the conserved Hip / type III secretion system gene hrcC // Eur. J. of Plant Pathology. 118 (3): 299-306 / et al.).

- DNA sequencing (determination and analysis of nucleotide sequences of fragments) / Sanger F. et al. 1977. Nucleotide sequence of bacteriophage phi X174 DNA //

Nature. 265 (5596): 687-95 /.

The third group of methods includes:

- AFLP and its various modifications (Vos, P. et al. 1995. AFLP: a new technique for DNA fingerprinting // Nucleic Acids Res. 23: 4407-4414; Savelkoul PHM, Aarts HJM, De Haas J., Dijkshoorn L. , Duim B., Otsen M., Rademaker JLW, Schouls L., Lenstra JA 1999. Amplified-Fragment Length Polymorphism Analysis: the State of an Art // Journal of clinical Microbiology. 37 (10): 3083-3091), in including saAFLP (a technique based on comparing the lengths of amplified fragments obtained by initially fragmenting the total body DNA with restriction enzymes, then doping the obtained restriction fragments with short (10-30 bp) double-stranded nucleotide sequences (adapters) and subsequent PCR with primers, nucleotide sequences that are complementary to adapter sequences);

- PCR-RFLP (a technique based on a comparison of the lengths of restriction fragments of target genes previously accumulated in high concentration by PCR) / Gurtler V., Wilson V.A., Mayall B.C. 1991. Classification of medically important clostridia using restiction endonuclease site differences of PCR-amplified 16S rRNA // J.Gen. Microbiol. 137: 2673-2679; Kostman J.R., Edlind T.D., LiPuma J.J., Stull T.L. 1992. Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reaction ribotyping // J. Clin. Microbiol. 30: 2084-2087 /.

Each of these methods has its advantages and disadvantages, however, their combined use allows us to ensure high reliability of identification and subsequent differentiation of taxonomic orders of different levels.

The prototype of our technique is a technique based on the amplification, sequencing and / or restriction of the intergenic region of the 16S pPHK and 23S rRNA ribosomal cluster, which allows one to simultaneously identify, identify and differentiate different taxa of prokaryotes / Jannes G., Rossau R., Van Heuverswyn H. 1995 Probes targeted to rRNA spacer region, methods and kids for using said probes, for the detection of respiratory tract pathogens. WO 96/00298 /. The method is quite universal, because almost all microorganisms have this intergenic region. However, its significant drawback is the high level of variability of the selected intergenic region, which requires the creation of a large collection of their sequences to create taxon-specific primers. However, this does not guarantee the absence of non-specific amplification with other taxa. It also makes it difficult to predict the operability of such primers on unknown representatives of the same taxon. Moreover, this intergenic region can be represented in the genome of the same organism in several copies that differ not only in length but also in the composition of the nucleotide sequence, which makes it very difficult to identify the carrier of such intergenic regions.

tRNA-PCR (tDNA-ILP) - PCR technique using consensus primers specific for the nucleotide sequences of bacterial, plant and animal tRNA genes / Welsh J., McClelland M. 1991. Genomic fingerprints produced by PCR with consensus tRNA gene primers // Nucleic Acids Res. 19: 861-866 /. Amplification takes place under fairly mild conditions: the primer annealing temperature is 50 ° C, the number of cycles is 40. The products of this reaction are usually separated in a polyacrylamide gel, and the sequencing of individual DNA fragments is carried out by means of cloning. In bacterial genomes, tRNA genes can be arranged in clusters consisting of several tandem repeating units / Green C. J., Void B. 1993. Staphylococcus aureus has clustered tRNA genes // J. Bacteriol. 175: 5091-5096; Void B. 1985. Structure and organization of genes for transfer ribonucleic acid in Bacillus subtilis // Microbiol. Rev. 49: 71-80 /. Such clusters - polycistrons - were found in Bacillus, Staphylococcus, Enterococcus, Lactobacillus, Streptococcus, Escherichia, Mycoplasma, Acinetobacter. However, not all bacteria have tRNA genes organized in clusters and it is not always possible to select consensus primers for them. So for the symbiotic bacteria of the genera Rhizobium, Sinorhizobium and phytopathogenic bacteria of the genus Xanthomonas, the tDNA-ILP approach is unacceptable, and most of the work on the application of this method is directed to clinical isolates of animals and humans / McClelland M., Petersen C., Welsh J. 1992. Length polymorphisms in tRNA intergenic spacers detected by using the polymerase chain reaction can distinguish streptococcal strains and species // J.din. Microbiol. 30: 1499-1504; Muto A., Andachi Y., Yuzawa H., Yamao F., Osawa S. 1990. The organization and evolution of transfer RNA genes in Mycoplasma capricolum // Nucleic Acids Res. 18: 5037-5043; Ehrenstein B., Bernards A.T., Dijkshoom L., Gemer-Smidt P., Towner K.J., Bouvet P.J.M., Daschner F. D., Grundmann H. 1996. Actinetobacter species identification by using tRNA spacer fingerprinting // J. Clin. Microbiol. 34: 2414-2420; Alexander S.M., Grayson T.N., Chambers E.M., Cooper L.F., Barker G.A., Gilpin M.L. 2001. Variation in the Spacer Regions Separating tRNA Genes in Renibacterium salmoninarum Distinguishes Recent Clinical Isolates from the Same Location // J. Clin. Microbiol. 39 (1): 119-128 /. Welsh and McClelland followed the path of creating universal primers for all tRNA genes present in the genomes of various pro- and eukaryotes, however, due to differences in the nucleotide composition, resulting in mild PCR conditions, the appearance of “random” products, it becomes impossible to analyze DNA samples in a mixture .

The reliability of tests based on nucleic acid analysis is significantly dependent on the sensitivity and specificity of the primers used. Therefore, the cornerstone of this kind of analysis is the identification of nucleotide sequences that are unique to the group of organisms of interest. Most of the nucleic acid tests described in the literature and / or commercially available tests are aimed at determining only one single organism in a biological sample. Due to the fact that most biological samples can usually contain a large number of diverse organisms, a large number of separate analyzes for each of them is required. This kind of approach is very time-consuming and requires large material and time costs. As a result, the number of tests actually carried out in most diagnostic laboratories to analyze a single sample is limited to only a few, aimed at identifying some of the most common organisms. Therefore, it would be very convenient to have a system capable of quickly, easily determining a large number of different organisms simultaneously, in one test tube. The greater the number of organisms that can be tested in the same analysis, the more cost-effective this test procedure will be.

The objective of the invention is a method for identifying a single microorganism or the simultaneous identification of various microorganisms in a sample.

The invention relates to nucleotide sequences of the hin region located between copies of the tRNA-Glu genes of the same name (Fig. 1), which are designed to determine microorganisms in a biological sample by amplification, restriction and / or sequencing, as well as nucleic acid primers designed to amplify the specified intergenic region of microorganisms in a biological sample. The present invention also relates to nucleotide sequences of new intergenic regions, on the basis of which similar primers can be obtained.

Qualitative characteristics of the hin region are: the number of amplified fragments, which reflects the number of copies of the tRNA-Glu gene, the length and sequence of DNA. Open reading frames (ORFs) may also be located in the nucleotide sequence of the hin region, i.e. least volatile DNA regions. Such an intergenic region is a relatively short (from about 200 to 1000 nucleotide pairs) unique piece of DNA that is present in the genome of proteobacteria in a single copy. The nucleotide sequence of the tRNA-Glu gene, as well as the number of copies thereof, are conservative at the generic level in a biological object, and sometimes groups of related genera. One of the most valuable advantages of the hin region is that its sequence is unique at the species level, which allows us to accurately determine the taxonomic position of the studied object (Fig. 2). The amplification products will be one, two and three DNA fragments of different lengths, which indicates the number of copies of the tRNA-Gly genes of the same name: two, three and four, respectively (Fig. 3). Thus, synthetic DNA molecules used as primers for a specific amplification reaction are selected for target sequences of tRNA-Gly genes of the studied genera of prokaryotes, which are available in the NCBI publicly available international genetic database (Table 1). For example, for bacteria of the genus Xanthomonas these are: 5'-ASASSASSSTTTSAS-3 'and 5'-CTAGACGATGGGGAC-3' for the genus Rhizobium these are: 5'-GGAAGAGGGGTTGGACT-3 'and 5'-AGAATGAAAATCTGGTG-3' and for the genus Sinorh - 5inh -CGATTCCCGTTGGGCGT-3 'and 5'-TTCCGCCGTGAAAGGGC-3' The aforementioned properties make it possible to create "primer panels" that simultaneously determine the set of organisms that may be present in a particular type of biological sample.

The proposed intergenic region (hin region) has a high resolution, allows identification of prokaryotic organisms, including closely related ones, up to the level of a group of strains, and also allows quick and efficient diagnosis of target biological objects. This is a valuable criterion for its use as a method for rapid identification of a biological object. The advantages of the Hin-region PCR method also include: greater informational content of the hin-region compared to analogues (16S rRNA, gyrB and other protein-coding genes), reproducibility and reliability of the results, simplicity and speed in execution, and the ability to automate the process.

In addition, being flanked by conserved nucleotide sequences of tRNA-Glu gene copies, all intergenic regions of the same (sometimes groups) genus can be simultaneously amplified using one pair of primers. On the other hand, based on the sequences of intergenic regions themselves, sets of specific primers or probes can be obtained that allow the detection of a prokaryotic organism at the species level or the level of a group of strains.

Sets of primers for the hin region allow identification of prokaryotes in which this sequence is flanked by 2 or more copies of tRNA-Glygen. Currently, at least the following groups or types of bacteria can be identified:

Thus, it is an object of the present invention to select taxon-specific primers or sets of such primers that target the hin region sequence and which can reliably determine and identify at least one and preferably more than one organism in a biological object.

It is also an object of the present invention to provide novel hin region sequences from which taxon-specific primers can be constructed.

In addition, the principal objective of the present invention is to select specific primers and sets of primers based on data from the hin region sequences that allow the determination and identification of specific panels of organisms belonging to a common taxon, or organisms possibly present in a sample of the same type.

The selection procedure usually consists of theoretical and experimental parts. First of all, different intergenic sequences must be compared with the corresponding sequences of "nearest neighbors" or intergenic sequences of other microorganisms possibly present in this sample. This requires the determination of the nucleotide sequence of the intergenic region. Based on this comparison, mismatch regions can be determined from which primers with the desired hybridization properties are prepared. In addition, the specificity and sensitivity of the probes must be checked on a large collection of strains belonging both to the identified taxon and to other taxa.

The nucleotide composition of the probe is significant because G-C base pairs have greater temperature stability compared to AT base pairs due to the presence of an additional hydrogen bond between the bases. Consequently, nucleic acid hybrids with a higher G-C content will be more stable at high temperatures.

A list of selected primers from the hin region sequences described in the present invention is shown in Table 1. The following definitions of terms and expressions used in various embodiments of the present invention are set forth.

The term “intergenic region” refers to a DNA sequence flanked by copies of tRNA genes of the same name.

The term "taxon" can refer to a complete genus or subgroup within a genus, species, or even an intraspecific variety (subspecies, serovars, patovars, biovars, etc.).

The term “nearest neighbor” means a taxon that is known or is assumed to be closest to the organism of interest in terms of DNA homology, and which must be distinguished from the organism of interest.

The term "specific amplification" or "specific primers" means that these primers amplify only the intergenic region of those organisms for which they were developed, but not other organisms.

The term "sensitivity" means the number of false negative results: i.e. a sensitivity of 99% suggests that out of 100 defined strains, 1 was skipped.

The term "specificity" means the number of false positives: i.e. 98% specificity suggests that for 100 determined strains, 2 appear to belong to organisms for which this test was not intended.

The term "resolution of the method" means the limit level of the taxon, below which the samples can no longer be distinguished using this method.

The term "reproducibility" means that the result obtained in a series of repetitions will be stable and the same.

The term "reliability" means the consistency of the results obtained by this method and alternative methods, while the results objectively reflect the properties of a biological object, and are not random in nature.

The term "primer" refers to a single-stranded oligonucleotide DNA sequence capable of acting as an initiation point for the synthesis of a nucleotide chain complementary to the copied DNA strand. The length of the primer and the nucleotide sequence must be such as to provide annealing followed by the synthesis of primer-extended products. Preferably, the primer contains 15-25 nucleotides. The specific length and sequence of the primer depends on the complexity of the DNA target under study, as well as on conditions for using the primer, such as temperature and ionic strength of the solution.

The term "Real-time PCR" means the amplification reaction with quantitative and qualitative determination of the products formed during it.

The amplification method used is a polymerase chain reaction / Saiki R., Gelfand D., Stoffel S., Scharf S., Higuchi R., Horn G., Mullis K., Erlich H. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase // Science. 239: 487-491 /.

A “sample” can be any biological material taken either from natural sources (soil, water, plant tissues) or obtained in laboratory conditions (pure cell cultures, including plant).

The determination and identification of the target material can be carried out using one of the sequencing methods described in the literature and known to specialists in this field.

Before studying new bacterial strains, it is necessary to conduct a “hin region of PCR” on a representative sample of already identified bacteria and compile a database of specific sequences for each species within the genus of interest.

Stages of the analysis:

1. Isolation of DNA from a biological object.

2. Selection of temperature and time conditions and PCR with a pair of primers PF and PR specific for this genus (taxon-specific primers at the genus level). To achieve high specificity of amplification, it is recommended to use Hot-start polymerase. To reduce the analysis time, it is possible to increase the instrumental rate of heating and cooling the reaction mixture by reducing its volume (to 1-2 μl) and transferring the process to a microtiter level with the possibility of detecting amplification products in real time.

3. Visualization of hin-region PCR products obtained during amplification in a 1.5% agarose gel, either using Real-time PCR, or using fragment analysis on genetic analyzers.

4. It is possible, but not necessary, to fragment the resulting products during PCR amplification of the hin region with restriction enzymes, preferably with a four-base recognition site, and subsequent electrophoretic separation of the restriction products in a 4.0% agarose gel.

5. Enzymatic purification of the PCR product using alkaline alkaline phosphatase (SAP) and Exonuclease I.

The reaction mixture: SAP - 0.05 u; 10 × SAP buffer - 0.35 μl; Exol - 0.5 units; PCR product - 10 μl; sterile water - up to 20 μl.

Temperature-time profile: 37 ° С - 30 min, 80 ° С - 15 min.

6. Direct sequencing with one of the taxon-specific primers.

7. Comparison of the obtained DNA sequence with the available database and drawing up a conclusion.

Example 1: Identification of phytopathogenic bacteria of the genus Xanthomonas

Xanthomonas spp. capable of causing disease in at least 124 species of monocotyledonous and 268 species of dicotyledonous plants, including the most important crops of the nightshade families, Asteraceae, Cruciferous, Grain and many others / Leyns F., Cleene M.D., Swings J.-G., Ley J.D. 1984. The host range of the genus Xanthomonas // Botanical review. 50: 308-356; Vauterin L., Swings J. 1997. Are classification and phytopathological diversity compatible in Xanthomonas // J. of Industrial Microbiol. and Biotech. 19 (2): 77-82 /. As a result of the introduction of industrial technology of crop production, including the use of hybrid seeds from international producers, the distribution of homogeneous genotypes of crops in different regions of the world, centralized seedling cultivation and mechanized processing of crops, the harmfulness of bacterioses increases and expands its distribution. The object of the study is complex: the taxonomy of the genus Xanthomonas seems controversial, since there is no explicit correspondence between the physiological and genetic properties of numerous clonal groups within this genus, and the relationships between different pathovariants within individual species are poorly understood. The very concept of a bacterial species differs from the general concept of a species in microbiology because the pathogenicity of bacteria of the genus Xanthomonas on similar plants does not prove their genetic proximity / Liao C.-H., Wells J.M. 1987. Association of pectolitic starins of Xanthomonas campestris with soft rot of fruits and vegetables at retail markets // Phytopatology. 77: 418-422 /. In this regard, the task of detecting, identifying and diagnosing these phytopathogens is becoming increasingly important.

The study of xanthomonad polymorphism by reference markers was carried out in great detail by Punina N.V., 2009 / Punina N.V., 2009. Assessment of the genetic diversity of phytopathogenic bacteria of the genus Xanthomonas sp. and the development of molecular markers for their diagnosis. Abstract. dis .... Cand. biol. sciences. Moscow. 27 p. /. According to the results of the study, the gene for gyrase B (gyrB) was recognized as the most promising taxonomic marker capable of competing with the “16S rRNA gold standard”. The phylogenetic tree, based on a comparison of its nucleotide sequences in 113 strains of xanthomonads, fully reflects the genetic diversity of the population of these phytopathogens (Fig. 53). However, groups of strains that were difficult to separate remained, and it was necessary to propose a technique for PCR fingerprinting at the intraspecific level.

The hin region was amplified using available genus-specific primers (forward primer: ACACCACCCTTTCAC, SEQ ID NO 1; reverse primer: TCTAGACGATGGGGAC, SEQ ID NO 2) using the polymerase chain reaction for the following species:

- X.campestris,

- X.euvesicatoria,

- X.vesicatoria,

- X.perforanse,

- X.gardneri,

- X.axonopodis,

- X.fuscans,

- X.arboricola

- X.albilineans

Sequencing of the intergenic region was performed using a method that dideoxy-terminating chain growth, determining the sequence of directly enzymatically purified PCR products combined with primers.

Figure 4-32 shows the nucleotide sequences of the intergenic regions of the various Xanthomonas species described above. The obtained nucleotide sequences were compared with each other and with sequences already known from the available data banks. The corresponding sequences are shown in Figs. 4-14. According to the picture obtained by sequencing, the tested strains could be assigned to one of the following species or species groups. The tested strains belonging to each group are presented in table 2.

A study of the nucleotide polymorphism of the hin region in 39 collection xanthomonad strains showed greater informational content than that of the gyrase B gene, while maintaining the topology of the phylogenetic tree (Fig. 54). Based on the nucleotide sequences of the intergenic region of Xanthomonas sp. it is possible to select and chemically synthesize a number of taxon-specific primers for each species or group of xanthomonad strains.

This result illustrates that the use of taxon-specific primers belonging to the intergenic region can be useful for differentiating different groups of strains belonging to the same species according to the results of classical identification methods, and that these primers can be used to identify and describe new species among xanthomonads. In this case, all strains of Xanthomonas spp. Can be determined using primers SEQ ID NO 1 and SEQ ID NO 2, while using taxon-specific primers for the genus Xanthomonas, it is possible to quantify each of the groups of strains in the sample, for example, on the platform Real-time PCR.

Example 2: Identification of symbiotic bacteria of the genus Rhizobium

It is known that nodule bacteria are divided into two large groups - fast and slow growing, differing in the nature of the metabolism of carbohydrates and nitrogen compounds, growth rates, etc. The first group includes bacteria of the genus Rhizobium, which includes rhizobia, characterized by wide specificity and the ability to infect various species of legumes (for example, Rhizobium leguminosarum, infecting peas, vetch, chinat, lentils, beans, clover and beans), as well as narrow specificity, for example, clover symbionts. This genus is of particular interest: having a complex taxonomic structure, due to frequent interspecific recombination between different alleles of taxonomically important genes (such as 16S rRNA, dnaK, nod genes), it is not always possible to adequately classify rhizobia isolates. This leads to the need for additional genetic data / Eardly B.D., Nour S.M., van Berkum P., Selander R.K. 2005. Rhizobial 16S rRNA and dnaK. genes: mosaicism and the uncertain phylogenetic placement of Rhizobium galegae // App.and Env. Mic. 71 (3): 1328-1335; Suominen L., Roos C., Lortet G., Paulin L., Lindström K. 2001. Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer // Mol. Biol. Evol. 18 (6): 907-916; Vinuesa P., Rojas-Jiménez K., Contreras-Moreira B., Mahna SK, Prasad BN, My H., Selvaraju SB, Thierfelder H., Werner D. 2008. Multilocus sequence analysis for assessment of the biogeography and evolutionary genetics of four Bradyrhizobium species that nodulate soybeans on the asiatic continent // App.and Env. Mic. 74 (22): 6987-6996 /. Currently, a combination of phenotypic, biochemical and genotypic methods is used to classify, identify and diagnose nodule bacteria. The most promising methods of study and diagnosis are molecular biological.

In order to create a quick and reliable method for identifying, differentiating, and studying the biodiversity of the population structure of nodule bacteria, a method for comparative analysis of the nucleotide sequences of their hin region was proposed. This approach allows us to quickly and efficiently assess the population for the prospect of using individual symbiotic microsymbiont – macrosymbiont pairs in agricultural practice and to describe the genetic diversity of the nodule bacteria population in order to diagnose new ones, passportize commercially valuable, and identify promising strains producing biologically active substances.

The hin region was amplified using available primers (forward primer: GGAAGAGGGGTTCGACT, SEQ ID NO 3; reverse primer: AGAATGAAAATCTGGTG, SEQ ID NO 4) using the polymerase chain reaction for the following species:

- R.leguminosarum (including bv.viceae, bv.trifolii, bv.phaseoli);

- R.phaseoli;

- R. giardinii;

- R..galegae.

The determination of the nucleotide sequence of the intergenic region was performed using a genetic analyzer.

Figures 33-52 show the nucleotide sequences of the intergenic regions of the various Rhizobium species listed above. The obtained nucleotide sequences were compared with each other and with sequences already known from the available data banks. The corresponding sequences are shown in Figs. 33-37. According to the picture obtained by sequencing, the tested strains could be assigned to one of the following species or species groups. The tested strains belonging to each group are presented in table 3.

The results of electrophoretic separation of hin-region PCR products of more than 60 strains of the rhizobia collection showed the difference in length of this taxonomic marker. Classes of the hin region (genotypes) were isolated on the basis of differences in the structure and nucleotide sequences of this region. Thus, on the basis of differences in the structure of the nucleotide sequences of the hin region of Rhizobium species, a total of 9 species groups were identified, 4 of which were inside the species R.leguminosarum (Table 4). By comparing the nucleotide sequences of the hin region of the studied and typical rhizobia strains, the division into genotypes (species groups) by the hin region was confirmed, which corresponded to modern ideas about the taxonomic structure of this genus.

Based on the nucleotide sequences of the intergenic region of strains of Rhizobium spp. a number of taxon-specific primers can be selected and synthesized for each group of strains of interest.

Using primers SEQ ID NO 3 and SEQ ID NO 4, all strains of Rhizobium spp. Can be determined, while using taxon-specific primers for the genus Rhizobium, one can quantify each of the groups of strains in a sample, for example, on the Real- time PCR, studying, for example, the composition of the natural population of soil microorganisms.

Claims (1)

A method for identifying and differentiating a prokaryotic microorganism containing a hin region, comprising isolating and / or concentrating DNA present in a sample, amplifying an intergenic region, visualizing and sequencing amplified products, and comparing the obtained amplification products and their nucleotide sequences with known ones, characterized in that the nucleotide sequence located between copies of tRNA-Glu genes, which is a the hin region, which is amplified using rhodospecific primers and sequenced, the identification and differentiation of prokaryotic microorganisms is carried out in this case on the basis of the number, length and nucleotide sequence of the products of such a reaction characterizing the hin region.

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