RU2514663C2 - Method of differentiating bacillus anthracis from other closely related species of genus bacillus based on determining differences in structure of chromosomal genes - Google Patents

Abstract

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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Description

The invention relates to the field of molecular microbiology, in particular to the diagnosis of anthrax pathogen. The proposed invention allows you to quickly and efficiently detect Bacillus anthracis DNA in biological material and environmental objects during diagnostic studies in practical health care and veterinary medicine and to reliably differentiate from other bacillary species.

The causative agent of anthrax is a gram-positive spore-forming microorganism belonging to the genus Bacillus. The closest phylogenetic relationship with B. anthracis consists of species B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis. The similarity of genetic, morphological and physiological characteristics allows us to distinguish them in a special taxonomic group, the so-called "1st group of bacilli."

The classical scheme for studying the material for the presence of the causative agent of anthrax is based on the determination of phenotypic signs — colony morphology, penicillin sensitivity in the pearl necklace test, sensitivity to diagnostic bacteriophages, the ability to form L-glutamine capsules on special media, and the absence of hemolysis on defibrated agar sheep blood, lack of mobility and activity of alkaline phosphatase. Nevertheless, quite often strains of B. anthracis are isolated, which differ in one or more features from a typical characteristic.

A known method of differentiation of strains of anthrax microbe by virulence in vitro, carried out on special nutrient media for testing the formation of capsules and anthrax toxin in a CO ₂ atmosphere [1]. A known method for the identification of B. anthracis with differentiation of strains by the production of capsules, protective antigen and S-layer antigens consists in seeding the strain culture on a special medium for testing capsule formation in a CO ₂ atmosphere and subsequent formulation of the immunodiffusion reaction using sera to a protective antigen and proteins of the S-layer of anthrax microbe [2]. However, these methods are designed to work with a pure culture of B. anthracis, with their help it is impossible to detect the causative agent of anthrax in biological material and environmental objects and differentiation from other representatives of the genus Bacillus.

The modern development of genetics and molecular microbiology allows the diagnosis of pathogenic bacteria based on amplification and sequencing technologies. Molecular genetic differentiation has a high resolution and excludes various interpretations associated with the phenotypic variability of bacteria. In addition to the chromosome, two plasmids, pXO1 and pXO2, are present in the genome of the causative agent of anthrax. Plasmid genes determine the synthesis of the main pathogenicity factors of the anthrax microbe. The presence of plasmids pXO1 and pXO2 is a differential diagnostic character that distinguishes B. anthracis from other representatives of the genus Bacillus. Almost all domestic and foreign PCR systems for the indication of the causative agent of anthrax are based on the identification of sequences of plasmid genes.

A known method for identifying the causative agent of anthrax using the commercial GenSib kit - “Test system for detecting B. anthracis pXO1 + DNA by the polymerase chain reaction” (FKUZ RosNIPCHI “Microbe”, Saratov) [3]. The kit contains primers complementary to the pagA gene sequence fragment located on the pXOl plasmid. However, this test system does not detect monoplasmid strains of B. anthracis containing only plasmid pXO2, or non-plasmid derivatives. In addition, it cannot be used to distinguish vaccine strains of B. anthracis from virulent ones.

A known method for identifying the causative agent of anthrax using the commercial kit “AmpliSens ^® B. anthracis - FRT: a set of reagents for detecting DNA of vegetative forms and spores of Bacillus anthracis in biological material and environmental objects by polymerase chain reaction (PCR) with hybridization-fluorescence detection in “real time” mode (Federal State Institution Central Research Institute of Epidemiology, Moscow) [4]. The test system is based on the identification of a fragment of the pagA gene localized in the plasmid pXO1 and the sarA gene located on the plasmid pXO2. However, it does not detect strains of anthrax microbe lacking plasmids. Bacterial plasmids are less stable genetic elements than chromosomal DNA, and can be eliminated at different frequencies. On the other hand, plasmids identical to pXO1 and pXO2 are found in individual strains of closely related B. anthracis bacillary species [5, 6, 7]. Some of these unusual strains are isolated from humans or animals with symptoms of anthrax-like illness.

Test systems containing primers for fragments of chromosomal genes have more specificity. However, the search for species-specific chromosomal loci is difficult due to the high homology of the chromosomal DNA of the causative agent of anthrax and individual representatives of the species B. cereus [8].

Known methods for detection of B. anthracis and intraspecific differentiation by plasmid composition, based on the integrated use of primers for fragments of pag, cya, lef genes located on plasmid pXO1, the cap gene located on plasmid pXO2, and the Ba813 chromosome sequence [9, 10]. However, the uniqueness of the Ba813 marker for the anthrax causative agent is called into question after the discovery of separate strains of B. cereus and B. thuringiensis containing the Ba813 sequence identical to the anthrax causative agent [11].

A known method for the identification and differentiation of B. anthracis using a multiplex amplification test system, including primers for fragments of plasmid genes cya and sarC, as well as the chromosomal sap gene encoding the protein of the S layer [12]. However, according to the data of full-genome sequences presented in the databases of the National Center for Biotechnological Information (NCBI), the nucleotide sequences of the sap gene in strains of B. anthracis and individual representatives of other species of the genus Bacillus are identical.

Qi Y. et al., Comparing the chromosomal sequences of bacillary strains, found in the anthrax microbe a synonymous replacement of one nucleotide in the rpoB gene encoding the β-subunit of RNA polymerase [13]. The prospects of using this marker for the differentiation of B. anthracis among phylogenetically related species of the genus Bacillus is shown. However, subsequently, when testing 319 bacillary strains, a B. cereus strain was identified with the rpoB gene sequence identical to that of B. anthracis [14].

Agaisse H. et al. in the process of comparative analysis of the sequenced genomes of B. anthracis and B. cereus revealed a single nucleotide polymorphism in the chromosomal gene of the pleiotropic regulator plcR [15]. The transcriptional regulator plcR is responsible for various cell functions related to the pathogenicity of the microorganism and survival in an aggressive environment. The nonsense mutation in the plcR gene is used in the typing of B. anthracis strains of various origins and belongs to the canonical SNP (single nucleotide polymorphism) [16]. Hurtle W. et al. revealed a single synonymous nucleotide substitution in the chromosomal gyrA gene of B. anthracis strains, which makes it possible to distinguish them from representatives of other bacillary species [17]. Based on the gyrA gene polymorphism, a probe with a fluorescent label was created. However, to identify genetic changes in the form of single nucleotide substitutions, special equipment is needed for real-time PCR or sequencing. In addition, mutations in the form of single nucleotide substitutions are subject to reverse reversion.

Irenge L. et al. Real-time two-step PCR has been proposed for the diagnosis of anthrax infection [18]. At the first stage, B. anthracis strains are screened using the plasmid genes of the anthrax microbe, and at the second stage, B. anthracis strains are more specifically identified using chromosome targets for the pur A and ptsI genes. However, a diagnostic study, carried out in two stages, is longer in time and less economical.

Wielinga P. et al. developed multilocus real-time PCR for the simultaneous detection of the chromosomal and plasmid loci of the causative agent of anthrax. Type 3 lambda prophage, which is part of the conservative region of B. anthracis DNA, was chosen as a chromosomal marker [19]. However, the probability of recombination events in the genome of B. anthracis, leading to the elimination of the prophage, is not ruled out.

In 2011, Ahmod N. et al. found a deletion of 72 bp in the conservative region of the chromosome of the causative agent of anthrax, in the gene ueaC encoding ATP synthase [20]. Testing with specific primers of 174 bacillary strains, including 39 anthrax pathogens, showed the presence of a mutation in the vast majority of strains of B. anthracis, with the exception of B. anthracis A1055. The UeaC chromosomal marker was recommended for differentiating the anthrax microbe from strains of other bacillary species by PCR and pyrosequencing. However, the use of a chromosomal mutation in the form of a deletion of part of the nucleotides in any gene does not allow the microorganism to be detected effectively. For the simultaneous indication of a bacterial pathogen and its differentiation from closely related species, the use of several species-specific loci is optimal.

The objective of the invention is to develop a method that allows to detect the causative agent of anthrax during diagnostic studies and to differentiate from other representatives of the genus Bacillus.

The technical result consists in an effective indication of the anthrax microbe and increasing the reliability of its differentiation from strains of phylogenetically related species.

The technical result is achieved by indicating the anthrax causative agent by the method of polymerase chain reaction with primers for chromosomal genes, which involves sample preparation, DNA isolation, PCR using synthetic oligonucleotide primers complementary to the sequences of the chromosomal genes fliC and hom2.

Primers for these genes have the following sequences:

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

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

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

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

Results are evaluated by the presence of amplified gene fragments and by their size. The formation of the amplification product with primers on a fragment of the fliC gene size of 153 bp indicates that the strain belongs to the species B. anthracis. The presence of an amplification product with primers on a 550 bp hom2 gene fragment, on the contrary, excludes the assignment of the isolate to the causative agent of anthrax.

The inventive method is based on the use of fragments of the chromosomal genes fliC and hom2 as target DNA. Earlier, in the process of comparative analysis of the nucleotide sequences of the life support genes of bacillary strains, we identified chromosomal mutations specific for the causative agent of anthrax. One of the highly specific sites was found in the fliC gene, which determines the synthesis of flagellin, the main component of the bacterial flagellum. At position 383 bp from the beginning of the fliC gene in strains of B. anthracis there is an insert of 231 nucleotides, leading to the cessation of flagellin synthesis. When stating PCR with primers complementary to the fragment of the sequences of the insert, the formation of the amplification product occurs upon interaction with B. anthracis DNA.

An additional DNA target for diagnostic studies is the mutation we found in the hom2 gene encoding the homoserine dehydrogenase enzyme. Deletion of 42 nucleotides in positions with 1042 bp 1083 bp each from the beginning of the hom2 gene, it blocks the synthesis of L-homoserine, from which methionine, threonine and cysteine are formed as a result of a series of successive reactions. Due to the relatively small extent of the DNA defect, the primers for the hom2 gene are selected so that the sequence of one of them falls directly on the deletion region. In this case, the formation of the amplification product is registered only in reactions with DNA of strains of bacillary species other than B. anthracis. The use of an additional chromosomal locus increases the reliability of differentiation of the causative agent of anthrax from strains of phylogenetically related species. The mutation in the hom2 gene, apparently, arose at the initial stage of branching from a common predecessor and the formation of B. anthracis as an independent species. In this case, it is a species-specific genetic marker.

The capabilities of the proposed method include the introduction of plasmid markers, which allows simultaneously with the detection of anthrax microbe to determine the accessory of the isolate to virulent, vaccine or atypical strains. The primers were calculated and the polymerase chain reaction conditions were selected taking into account the prospects of using primers for fragments of the chromosome genes fliC, hom2 and plasmid determinants pagA, cap A in one reaction mixture [19]. The size of the amplification products formed in multilocus PCR allows us to distinguish them from each other by electrophoretic separation in agarose gel. One-stage PCR is economically feasible and requires less time to conduct a study.

The method is as follows:

Isolation of DNA from material previously disinfected according to standard methods is carried out in accordance with MU 1.3. 2569-09 “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of the 1st-4th pathogenicity groups” (M., 2009).

The polymerase chain reaction is carried out according to the standard method (MU 1.3. 2569-09) using the above primers for the hom2 and fliC genes.

Oligonucleotide primers used for PCR amplification of fragments of the hom2 and fliC genes were calculated based on the nucleotide sequences of these genes in the bacillary strains presented in the 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. megaterium QM B1551, DSM 319; B. subtilis subsp.spizizenii, BSU35150, BSn5. Primers were analyzed using the BLAST computer program on the NCBI web server (http://www.ncbi.nlm.nih.gov/BLAST/) to confirm their specificity and lack of homology with nucleotide sequences of strains of bacillary species present in the databases (GenBank , EMBL, DDBJ).

Using the calculated primers, fragments of the hom2 and JliC genes are amplified in PCR, and the presence and size of the resulting amplicons are determined. Primers for the hom2 and fliC genes have the following sequences:

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

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

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

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

The polymerase chain reaction with amplification of fragments of the hom2 and fliC genes is carried out according to the following program: one cycle of 94 ° C for 5 min, 30 cycles at 94 ° C for 30 sec, 58 ° C for 30 sec, 72 ° C for 30 sec and final cycle - 3 min at 72 ° C. Electrophoretic analysis of PCR products is carried out by electrophoresis in a 2% agarose gel relative to the standard molecular weight markers with sizes from 100 to 3000 bp

For strains of anthrax microbe, the PCR product corresponds to the size of 153 bp, for strains of other bacillary species - 550 bp

Thus, the developed method allows for a short time to detect the causative agent of anthrax, as well as to conduct its reliable differentiation from other representatives of the genus Bacillus.

The invention is illustrated by example.

Example. Study of material containing a culture of strain B. anthracis or a strain of another bacillary species (Model experiment).

The disinfection of the test samples is carried out in accordance with the Methodological guidelines of MU 3.5.5.1034-01 "Disinfection of the test material infected with bacteria of pathogenicity groups I-IV during PCR."

DNA is isolated using the “Reagent Kit for Sample Preparation (DNA Isolation)” produced by the Federal Microbiological Research Institute of Microbiology.

The reaction mixture for the PCR reaction is prepared according to the recipe:

deionized water - 12.5 μl,

dNTP solution (2.5 mmol) - 2 μl,

primer fliC-F (12 pM / μl) - 0.5 μl,

primer fliC-R (12 pM / μl) - 0.5 μl,

hom2-F primer (12 pM / μl) - 0.5 μl,

hom2-R primer (12 pM / μl) - 0.5 μl,

NPF reaction buffer (× 10) - 2.5 μl,

MgCl ₂ 0.25 M - 0.8 μl,

Taq polymerase enzyme (5 units / μl) - 0.2 μl,

test DNA sample - 5 μl.

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

Amplification of fragments of the fliC and hom2 genes in PCR is carried out according to the scheme: denaturation - 94 ° C for 5 min, 30 cycles at 94 ° C - 30 sec, annealing of primers - 58 ° C - 30 sec, chain elongation 72 ° C - 30 sec and the final cycle of 3 minutes at 72 ° C. Electrophoretic analysis of PCR products is carried out in a 2% agarose gel.

In all samples containing B. anthracis DNA, an amplification product of 153 bp is synthesized, which determines the insert in the fliC gene. When testing the DNA of representatives of various bacillary species, an amplification product of 550 bp was detected, revealing unchanged hom2 gene sequences. Due to the deletion of part of the nucleotide sequences of the hom2 gene in anthrax pathogen strains, amplification with calculated primers for its fragment does not occur in these samples (drawing). The drawing shows the results of PCR analysis with primers for fragments of the fliC and hom2 genes of strains of B. anthracis and other bacillary species: 1. B. anthracis Pasteur; 2. B. anthracis 55; 3. B. anthracis Ihtiman; 4. B. anthracis KM86 (7); 5. B. anthracis KM86 (8); 6. B. anthracis Sterne 34F2; 7. B. anthracis STI-1; 8. B. anthracis KM35; 9. B. anthracis 29; 10. B. anthracis KM 91; 11. molecular weight marker "O '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. B. 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.

The sensitivity of the amplification reaction with primers fliC-F, fliC-R, hom2-F and hom2-R was evaluated by studying DNA samples isolated from ten-fold dilutions of the pure culture of anthrax pathogen B. anthracis, and amounted to 1 × 10 ² -1 × 10 ⁴ spore / ml.

Literature

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Claims (1)

A method for differentiating the anthrax causative agent from other closely related species of the Bacillus genus based on determining differences in the structure of chromosomal genes, which includes sample preparation, DNA isolation, PCR staging, characterized in that oligonucleotide primers complementary to the sequences of chromosomal genes having the following fliC and hom are used in PCR 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 amplification products, in which the formation of an amplification product of 153 bp indicates the belonging of the studied strain to the species B. anthracis, the formation of an amplification product of 550 bp in size indicates the belonging of the studied strain to another species of the genus Bacillus.

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