Method of differentiating bacillus anthracis from other closely related species of genus bacillus based on determining differences in structure of chromosomal genes. RU patent 2514663.

FIELD: biotechnology.

SUBSTANCE: method includes sample preparation, DNA isolation, PCR statement. At that in carrying out PCR, the oligonucleotide primers are used, which are complementary to sequences of the chromosomal genes fliC and hom2, having the following sequences: fliC-F: 5'-TGGAGCAGTAACAATTGG-3', fliC-R: 5'-GCACCACTGATAGAAATGTTAG-3', hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3', hom2-R: 5'-CACCAATTTCGTCTTTTACA-3', followed by electrophoretic analysis of the amplification products, when the formation of the amplification product is 153 bps in size it is indicative of belonging of the strain under study to the species B.anthracis, formation of the amplification product with size of 550 bps is indicative of belonging of the strain under study to the other species of the genus Bacillus.

EFFECT: invention enables to differentiate effectively the bacillus anthracis from other species of bacilli.

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The invention relates to the field of molecular Microbiology, in particular to the diagnosis of anthrax germs. The proposed invention allows to quickly and effectively identify DNA Bacillus anthracis in biological material and the environment when conducting diagnostic studies in practical health care and veterinary and implement a reliable differentiation from other bacillary species.

The causative agent of anthrax - gram-positive spore-forming organism belonging to the genus Bacillus. In most close phylogenetic relationship with Century anthracis are the types of B. cereus, B. thuringiensis, B. mycoides, Century pseudomycoides Century and weihenstephanensis. The similarity of the genetic, morphological and physiological characteristics allows to allocate them in a particular taxonomic group, the so-called "1-St group bacilli".

The classical scheme of research material on the presence of anthrax based on the definition of the phenotypic characteristics - colony morphology, sensitivity to penicillin in the test "pearl necklace", sensitivity to diagnostic bacteriophages, ability to formation of L-glutamine capsules for special environments, lack of hemolysis on agar with defibrinirovannaya the blood of sheep, lack of mobility and alkaline phosphatase activity. However, quite often isolated strains Century anthracis, which on one or more characteristics different from the typical characteristics.

The known method of differentiation strains of Bacillus anthracis in virulence in vitro carried out on a special nutrient medium for testing education capsules and anthrax toxin in the atmosphere FROM 2 [1]. The known method of identification Century anthracis with differentiation strains on production of capsules, Pro-tektivnye antigen or antigens S-layer consisting in sowing culture of strain on a special testing environment for education capsules in the atmosphere FROM the 2nd and subsequent formulation of reaction immunodiffusion using serums to the protective antigen and protein S-layer Bacillus anthracis [2]. However, these methods are designed to work with a pure culture Century anthracis, they cannot be used to detection of anthrax germs in biological material and environmental objects and differentiation from other representatives of the genus Bacillus.

Modern development of genetics and molecular Microbiology allows the diagnostics of pathogenic bacteria based amplification and sequencing technologies. Molecular-genetic differentiation has a high resolution and excludes various interpretations associated with phenotypic variability of bacteria. In the genome anthrax, in addition chromosomes, there are two plasmids - pXO1 and pXO2. Plasmid genes determine a synthesis of the main determinants of pathogenicity Bacillus anthracis. The presence of plasmids pXO1 and pXO2 a differential diagnostic feature that distinguishes a Century anthracis from other representatives of the genus Bacillus. Practically all domestic and foreign PCR display system anthrax based on detection of sequences plasmid genes.

There is a method of identifying anthrax using a commercial kit Gensis - "Test-system for detection of DNA Century anthracis Rho+by polymerase chain reaction" (FCUS antiplague research Institute "Microbe", Saratov) [3]. The set contains the primers complementary to the movie sequences of the gene pagA, located on a plasmid pXOl. However, this test system will not detect mikoplazmenne strains Century anthracis, containing only plasmid pXO2, or besplatnye derivatives. In addition, it is impossible to distinguish vaccine strains Century anthracis from virulent.

There is a method of identifying anthrax using a commercial kit "AmpliSens ® Century anthracis - FRT: reagent kit for detection of DNA vegetative forms and spores of Bacillus anthracis in biological materials and environmental objects by the method of polymerase chain reaction (PCR) with hybridization-fluorescent detection in "real time" (fsri Central research Institute of epidemiology, Moscow) [4]. The test system is based on the identification of the fragment pagA gene is localized in the composition of plasmids pXO1, and Sarah a gene located on a plasmid pXO2. However, it does not define the strains of Bacillus anthracis, deprived of plasmids. Bacterial plasmids less stable genetic elements than chromosomal DNA, and with different frequency to eliminiruyutza. On the other hand, plasmids, identical pXO1 and pXO2, found in the separate strains closely related Century anthracis bacillary species [5, 6, 7]. Some of these unusual strains isolated from humans or animals with symptoms resembling anthrax.

Greater specificity have a test system with primers on fragments of chromosomal genes. However, the search for species-specific chromosomal loci hampered by high homology chromosomal DNA anthrax and individual members of a species B. cereus [8].

Known methods of detection Century anthracis and intraspecific differentiation by plasmid composition, based on the integrated use of primers for gene fragments pag, cya, lef, located on a plasmid pXO1, cap gene, located on a plasmid pXO2, and chromosomal sequence VA [9, 10]. However, the uniqueness of the marker VA for anthrax questioned after the discovery of individual strains of B. cereus and B. thuringiensis containing a sequence We identical with anthrax [11].

The known method of identification and differentiation Century anthracis using multiplex amplification test systems, including primers on fragments of plasmid genes of Mas and SARS, as well as chromosomal gene sap encoding protein S-layer [12]. However, according to genome-wide sequences presented in the databases of the National Center for Biotechnology Information (NCBI), the nucleotide sequence of the gene sap in strains Century anthracis and certain other species of the genus Bacillus identical.

Y. Qi et al., comparing chromosomal sequences smear-positive strains found in Bacillus anthracis synonymous substitution of one nucleotide in the rpoB gene encoding β-subunit of RNA polymerase [13]. Perspectivity of use of this marker for the implementation of differentiation Century anthracis among phylogenetically close species of the genus Bacillus. But later when testing 319 smear-positive strains identified a strain of B. cereus with the rpoB gene sequence identical to a similar sequence in B. anthracis [14].

Agaisse N. et al. in the process of comparative analysis of sequenced genomes Century anthracis and B. cereus revealed polymorphism of single nucleotide in chromosomal gene pleiotropic regulator plcR [15]. Transcriptional regulator plcR responsible for various cell functions related to the pathogenicity of the organism and survival in a hostile environment. Nonsense mutation in the gene plcR used when typing of strains Century anthracis different origin and refers to the canonical SNP (single nucleotide polymorphism) [16]. Hurtle W. et al. revealed a single synonymous nucleotide substitution in chromosomal gyrA gene strains Century anthracis to differentiate them from representatives of other bacillary types [17]. On the basis of gyrA gene polymorphism was created probe with fluorescent label. However, to detect genetic changes in the form of single nucleotide substitutions requires special equipment for PCR in real time or sequencing. In addition, mutations in the form of single nucleotide substitutions subject reverse reverse.

Irenge L. et al. was offered a two-stage real-time PCR for the diagnosis of anthrax infection [18]. At the first stage of screening strains Century anthracis using plasmid genes of Bacillus anthracis, the second stage consists of more specific identification of strains Century anthracis with the use of chromosomal target genes And pur and ptsI. However, the diagnostic study carried out in two stages, with more time and less economical.

Wielinga P. et al. developed multiloci real-time PCR for the simultaneous detection of chromosomal and plasmid loci of anthrax germs. As chromosome marker selected lambda Profi 3 type that is part of a conservative region of DNA Century anthracis [19]. However, it is not excluded the probability of recombination events in the genome Century anthracis, leading to the elimination of propaga.

In 2011 Ahmod N. et al. find a deletion 72 P.N. in a conservative region of a chromosome anthrax, encoding ATP synthase gene weas [20]. Testing with specific primers 174 smear-positive strains, including 39 strains of anthrax, showed the presence of mutations in the vast majority of strains Century anthracis, except Century anthracis A. Chromosome marker UAAS was recommended for differentiation Bacillus anthracis from smear-positive strains of other types of PCR and persecutione. However, the use of chromosome mutations in the form deletions part of nucleotides in any gene does not allow to detect the microorganism. For simultaneous indication of bacterial pathogen and its differentiation from closely related species optimal use of several species-specific loci.

The objective of the invention is development of a method that allows detection of the causative agent of anthrax while conducting diagnostic studies and conduct differentiation from other representatives of the genus Bacillus.

The technical result consists in the efficient display Bacillus anthracis and improving the reliability of its differentiation from strains phylogenetically close species.

The technical result is achieved indication anthrax by polymerase chain reaction primers on chromosomal genes, which provides for sample preparation, DNA detection, staging PCR using synthetic oligonucleotide primers complementary sequences of chromosomal genes fliC and hom2.

Primers on these genes have the following sequence:

fliC-F: 5'-TGGAGCAGTAACAATTGG-3',

fliC-R: 5*-GCACCACTGATAGAAATGTTAG-3',

hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3',

hom2-R: 5'-CACCAATTTCGTCTTTTACA-3*.

The proposed method is based on the use of DNA targets fragments of chromosomal genes fliC and hom2. Earlier in the process of comparative analysis of nucleotide sequences of genes life support smear-positive strains we have identified specific to anthrax chromosomal mutations. One of the highly specific sites had been found in the gene fliC determining the synthesis of flagellin - the main component of the bacterial flagellum. In position 383 P.N. from the beginning of the gene fliC in strains Century anthracis is present insert 231 nucleotide, leading to the termination of the synthesis of flagellin. When setting PCR with primers complementary to the movie sequences insertion, education product amplification occurs in the interaction with DNA Century anthracis.

Additional DNA target for diagnostic studies serves as we found a mutation in the gene hom2, encoding the enzyme gomoserinlaktonazy. Deletion 42 nucleotides in the positions form 1042 by P.N. 1083 P.N. from the beginning of the gene hom2 blocks the synthesis of L-homoserine, from which a series of consecutive reactions are formed methionine, threonine and cysteine. Given the relatively small extent of the defect DNA primers for gene hom2 selected so that the sequence of one of them had directly on the area of the deletions. In this case, the education of product amplification is registered only in reactions with DNA strains bacillary species other than Century anthracis. Use extra chromosomal locus increases the reliability of differentiation anthrax from strains phylogenetically close species. A mutation in a gene hom2 apparently occurred at the initial stage of branches from common predecessor and the formation of a Century anthracis as a separate species. In this case it is species-specific genetic marker.

The possibilities of the proposed method, introduce a plasmid token that allows simultaneous detection of the anthrax microbe to determine the facilities to isolate virulent vaccine or atypical strains. Calculation of primers and selection conditions polymerase chain reaction conducted with a view to future use in one of the reaction mixture primers on the fragments of chromosomal genes fliC, hom2 and plasmid determinants pagA, cap And [19]. The size of the generated in multiloci PCR amplification products to separate them from each other in an electrophoretic separation in agarose gel. Staging a one-step PCR economically it is more efficient and requires less time to study.

The method is as follows:

DNA isolation from material previously decontaminated according to the standard procedure is carried out in accordance with MU 1.3. 2569-09 "Organization of work of laboratories, using the methods of amplification of nucleic acids when working with material containing microorganisms 1 - IV groups of pathogenicity (M, 2009).

Polymerase chain reaction carry out according to standard method (MU 1.3. 2569-09) applying the above mentioned primers on genes hom2 and fliC.

Oligonucleotide primers used for PCR amplification of the gene fragments hom2 and fliC, calculated on the basis of the nucleotide sequences of these genes have smear-positive strains presented in NCBI databases: B. anthracis Ames, Ames 0581, Steme, CDC 684, A0248; B. cereus ATCC14579, ATCC10987, ZK, AH187, B4264, AH820, G9842, Q1, 03BB102; Bacillus thuringiensis 97-27, Al Hakam, BMB171; B. weihenstephanensis KBAB4_4052, B. licheniformis ATCC 14580, DSM13; B. B. megaterium QM B1551, DSM 319; B. subtilis subsp.spizizenii, BSU35150, BSn5. Primers analyzed using computer programs BLAST on the web server NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) to confirm their specificity and the absence of homology with nucleotide sequences of smear-positive strains of the species present in databases (GenBank, EMBL, DDBJ).

Using calculated primers spend PCR amplification of the gene fragments hom2 and JliC determine the existence and the dimensions of the resulting amplicons. Primers on genes hom2 and fliC have the following sequence:

fliC-F: 5'-TGGAGCAGTAACAATTGG-3',

fliC-R: 5'-GCACCACTGATAGAAATGTTAG-3',

hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3',

hom2-R: 5'-CACCAATTTCGTCTTTTACA-3'.

Polymerase chain reaction amplification of the gene fragments hom2 and fliC perform the following program: one cycle 94 OC for 5 min, 30 cycles at 94 C is 30 sec, 58 degrees C - 30 s, 72 C is 30 seconds and the final cycle - 3 min at 72 C. Electrophoretic analysis of PCR products is carried out by the method of electrophoresis in 2% agarose gel relatively standard markers molecular masses with the size from 100 to 3000 gel

For strains of Bacillus anthracis, the PCR product corresponds to the size of 153 gel, for smear-positive strains of other species - 550 P.N.

Thus, the developed method will allow you to detect anthrax, as well as to conduct its reliable differentiation from other representatives of the genus Bacillus.

The invention is illustrated by example.

Example. The study material containing culture strain Century anthracis or strain another bacillary form (Modelling experiment).

Disinfection of samples tested carried out according to the Methodological instructions MU 3.5.5.1034-01 "Disinfection of the investigated material infected with bacteria of I-IV groups of pathogenicity, working by PCR".

DNA isolation is performed with the help of a Set of reagents for sample preparation (DNA)" produced FKUS antiplague research Institute "Microbe".

The reaction mixture for the production of PCR reactions prepare for the recipe:

water deionized - 12,5 MKL

the solution dNTP (2.5 mm) - 2 MKL

primer fliC-F (12 gr/ ml) - 0,5 MKL

primer fliC-R (12 gr/ ml) - 0,5 MKL

primer hom2-F (12 gr/ ml) - 0,5 MKL

primer hom2-R (12 gr/ ml) - 0,5 MKL

the reaction buffer NPF (x 10) - 2,5 MKL

MgCl 2 0.25 M - 0,8 MKL

the enzyme Taq polymerase (5 units/ ml) - 0.2 to MKL

the sample DNA - 5 mm.

The total volume of the reaction mixture is 25 MKL 1 the sample.

Amplification of the gene fragments fliC and hom2 in PCR performed according to the scheme: denaturation - 94 OC for 5 min, 30 cycles at 94 C is 30 sec, the annealed primers - 58 degrees C - 30 sec, elongation of the chain 72 C is 30 seconds and the final cycle - 3 min at 72°N Electrophoretic analysis of PCR products carry a 2% agarose gel.

In all samples containing DNA Century anthracis, synthesized product amplification size 153 P.N. that defines the insertion of a gene fliC. When DNA testing of representatives of various bacillary species register the product amplification a 550 P.N. detecting unmodified gene sequence hom2. Due to the deletion of part of the nucleotide sequences of the gene hom2 in strains of anthrax amplification with calculated primers on its part in these samples is not happening (the drawing). The drawing shows the results of PCR-analysis with primers for gene fragments fliC and hom2 strains Century anthracis and other bacillary forms: 1. Century anthracis Pasteur; 2. Century anthracis 55; 3. Century anthracis Ihtiman; 4. Century anthracis KM (7); 5. Century anthracis KM (8); 6. Century anthracis Sterne 34F2; 7. Century anthracis STI-1; 8. Century anthracis KM; 9. Century anthracis 29; 10. Century anthracis 91 KM; 11. a token of molecular masses "On' GeneRuler™ 100bp DNA Ladder Plus redy-to-use" (Fermentas) (3000, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500,400, 300, 200, 100 P.N.); 12. Century thuringiensis HD-1; 13. B. thuringiensis 2090; 14. B. thuringiensis 69/6; 15. B. cereus 569; 16. B. cereus 108; 17. B. cereus 8; 18. B. pseudoanthracis 104.

Sensitivity amplification reaction with primers fliC-F, fliC-R, hom2-F and hom2-R was estimated in the study of samples of DNA extracted from a ten-fold dilutions of pure cultures of anthrax Century anthracis, and amounted to 1 x 10 2 -1 x 10 4 spores/ml

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Method of differentiation anthrax from other closely related species of the genus Bacillus on the basis of differences in the structure of chromosomal genes, providing for sample preparation, DNA detection, staging PCR, wherein by PCR apply oligonucleotide primers complementary sequences of chromosomal genes fliC and hom2 with the following sequence: fliC-F: 5'-TGGAGCAGTAACAATTGG-3', fliC-R: 5'-GCACCACTGATAGAAATGTTAG-3', hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3', hom2-R: 5'-CACCAATTTCGTCTTTTACA-3' followed electrophoretic analysis of amplification products, in which the formation of product amplification size 153 P.N. shows toiletries investigated strain to mind Century anthracis, education product amplification of a 550-BP suggests the facilities studied the strain another species of the genus Bacillus.