Method of differentiation of strains of principal and nonprincipal causative plague agent and pseudotuberculosis agent by polymerase chain reaction

FIELD: medicine.

SUBSTANCE: method provides recovery of DNA of an analysed strain followed by PCR with using nucleotide primers of terC, ilvN and inv genes of the following sequences: 89-S - AATCAAATCTCGCCCAGC, 89-As -GCTGCGTATCATTTCACC; 45-S - AGTGGTCTGCTTCTCTGG, 45-As -CGGCATACACAGAATACC; inv839 - TACCTGCACTCCCACAAC, inv1007 -CCCATACGCTGATCTACC. The analysed strains are differentiated by matching the sizes of the derived fragments of terC, ilvN and inv genes with the similar fragments in typical strains of principal and nonprincipal plague agents.

EFFECT: invention allows quick, effective and reliable differentiation of the strains.

1 tbl, 3 ex

 

The invention relates to the field of medical Microbiology, in particular to subspecific differentiation of strains of the plague pathogen and differentiation of plague and pseudotuberculosis microbe.

Plague pathogen which is a type of bacteria, Yersinia pestis, still poses a real threat to human health associated with existence on different continents numerous active natural foci of plague, and the use of the plague pathogen in bioterroristic acts. Every year in the world there are a sufficiently large number of cases of plague, which often lead to death. Because of this importance is the development of accurate methods of detection Y.pestis and differentiation of this agent from other bacteria, in particular from the closely related Yersinia pseudotuberculosis. The look of Y.pestis heterogeneous in composition and is divided into the main subspecies subspecies (ssp) pestis and group non-core subspecies: Altai - ssp.altaica, Caucasian - ssp.caucasica, wagasky - ssp.ulegeica, Hissar - ssp.hissarica. The subspecies of the plague pathogen differ in virulence and epidemiological significance. The strains of the main subspecies are characterized by high virulence and high epidemic potential. Strains of non-core races have selective virulence, epidemically insignificant and hardly cause for which olivani person. In this regard, along with the need for differentiation of plague and pseudotuberculosis microbe is not less important and differentiation of subspecies Y.pestis, and, in particular, the detection of these strains of the main subspecies. The development of efficient and reliable methods for rapid identification of the main subspecies of Y.pestis among closely related pathogenic Yersinia has important practical value.

Currently, the differentiation of strains of the plague pathogen main and non-main subspecies carried out taking into account the different expression of a number of biochemical characteristics: the fermentation of monosaccharides - ramnose, melibiose, products isocitrate and others. However, the phenotypic manifestation of the plague pathogen properties depends on the conditions of cultivation of strains, the quality of the environments, and the isolation of the pathogen in pure form, which greatly complicates and increases the time of analysis.

Greater efficiency and speed of obtaining results different method of polymerase chain reaction (PCR), based on the use of genetic characteristics of strains of various bacteria. For PCR does not necessarily isolation of pure cultures of the pathogen, since the method is highly sensitive and allows the determination of species and subspecies belonging to b is Cherie if you have a small number of cells in the material of the test sample.

Up to the present time PCR was used for detection of the causative agent of plague, as well as to differentiate it from the causative agent of pseudotuberculosis, but almost never used it to differentiate between main and non-main subspecies of Y.pestis. For detection of the causative agent of plague in PCR used the nucleotide sequences of various genes caf, pla, lcrV, 3a, irp-2 (Neubauer H. et al., 2000; Rahalison L. et al., 2000; Hongwei Z.J.L., 2000; Balakhonov S.V. with coawt; Garanina S.B. et al., 2001; Knots YU et al., 2001; L. Radnedge et al., 2001; Savostin, H.E., 2004; Kuske C.R. et al., 2006; Kuklev B.E. et al. 2006).

So, there is a method of detecting bacteria of the genus Yersinia and differentiation of a human pathogenic species of Yersinia by polymerase chain reaction (patent RU 2354700 published 10.05.2009, on the basis of sequence portion of a gene ompF Porin. This method allows you to split the pathogenic species of Yersinia - causative agents of plague and pseudotuberculosis, intestinal yersiniosis at the species level, but it does not allow for identification of Y.pestis strains of main and non-main subspecies.

Known another method for rapid detection and differentiation of Y.pestis and Y.pseudotuberculosis (DE 10124342 (A1), published 28.11.2002 g), which provides a two-step detection of the DNA of the causative agents of plague and pseudotuberculosis: PCR and hybridization with specific probes, which increases n is dejnost and efficient way. However, this method also does not allow to differentiate strains of Y.pestis main and non-main subspecies.

Closest to the proposed invention, the essential features is the way of differentiation of the plague and pseudotuberculosis microbe with simultaneous intraspecific differentiation of strains of the plague microbe (EN 2332464 published, 27.08.2008), which is based on the use of the variability of the nucleotide sequence of a segment of a chromosome hutG - YP01967. However, the disadvantage of this method is that the plot hutG - YP01967 is located in the unstable region of the genome Y.pestis, which can often be lost in strains of the main subspecies, which makes it impossible for the differentiation of these strains using the proposed method. In addition, this method is difficult to interpret, requires the use of a large group of reference strains, and also has some error, because it does not allow to avoid coincidence of the sizes of PCR fragments from some strains of the main and non-main subspecies, which requires additional research (e.g., sequencing) to determine the subspecific affiliation of strain. Other publications about the use of the PCR method to differentiate strains of the plague pathogen main and non-main subspecies in the literature are absent, as are absent, and given what's about the parts of the genome Y.pestis, the use of which would allow to quickly and efficiently perform such differentiation.

The objective of the invention is to develop a simple interpretation, reliable and rapid method for the differentiation of strains of the plague pathogen main and non-main subspecies and strains of the causative agent of pseudotuberculosis by PCR method.

The technical result consists in increasing the efficiency of differentiation of strains of the plague pathogen main and non-main subspecies with simultaneous differentiation of strains of the causative agent of pseudotuberculosis.

The inventive method is based on using as a DNA target of chromosomal genes livelihoods terC, ilvN and inv, encoding respectively the protein resistance tellurium, acetolactate and invasin-adhesin.

The technical result is achieved by the method of differentiation of strains of the plague pathogen main and non-main subspecies and the causative agent of pseudotuberculosis by polymerase chain reaction, which involves the isolation of the DNA of strain, carrying out polymerase chain reaction with amplification of the gene fragments terC, ilvN and inv investigated strain, while the primers for these genes have the following sequence:

89-S - AATCAAATCTCGCCCAGC,

89-As - GCTGCGTATCATTTCACC;

45-S - AGTGGTCTGCTTCTCTGG,

45-As - CGGCATACACAGAATACC,

inv839 - TACCTGCACTCCCACAAC,

inv1007 - CCCATACGCTGATCTACC,

and dimensioning am limitirovany fragments with subsequent differentiation of the studied strains, which is carried out by comparing the sizes of the obtained gene fragments terC, ilvN and inv with similar fragments in the typical strains of the plague of the main and non-main subspecies.

The method is as follows.

The isolation of the DNA of the studied strains of the plague microbe performed by standard methods in accordance with MU 1.3.1794-03 "Organization of work in the research by PCR material infected by microorganisms 1-11 of pathogenicity groups.

Polymerase chain reaction carried out according to standard methods (MU 1.3.1794-03) using the above primers for genes terC, ilvN and inv. Oligonucleotide primers used for amplification in PCR fragments terC, ilvN and inv, calculated on the basis of the nucleotide sequences of these genes in strains of the plague microbe Y. pestis CO92, KIM, Antiqua, Nepal516, Angola, 91001, Pestoides F and Y. pseudotuberculosis IP31758, presented in the database of NCBI GenBank. Using designed primers is carried out in PCR amplification of gene fragments terC, ilvN and inv and determines the size of the obtained gene fragments. Primers for the genes terC, ilvN and inv have the following sequence:

89-S - AATCAAATCTCGCCCAGC,

89-As - GCTGCGTATCATTTCACC;

45-S - AGTGGTCTGCTTCTCTGG,

45-As - CGGCATACACAGAATACC,

mv839 - TACCTGCACTCCCACAAC,

invl007 - CCCATACGCTGATCTACC.

Polymerase chain reaction with amplification of the gene fragments terC, ilvN and inv carry out the following program: cycle of 94°C for 5 min, then 35 cycles at 94°C 45 sec, 55°C 1 min, 72°C 45 s and a final cycle of 3 min at 72°C. the sizing educated in PCR fragments of genes terC, ilvN and inv in the tested strains carried out by electrophoresis in 3% agarose gel relative to the standard markers molecular masses ranging in size from 100 to 1000 BP We found that the typical strains of the plague pathogen main subspecies are formed in PCR fragments of genes terC - 300 gel, ilvN - 515 P.N. and inv - 877 P.N.; minor strains of the subspecies accordingly, 389, 560 and 877; and the strains of Yersinia pseudotuberculosis microbe - 389, 560 and 169 (table). Further differentiation of the studied strain is carried out by comparison with the data presented in the table.

The invention is illustrated by examples.

Example 1. Differentiation of strain No. 1 (Model experiment).

The isolation of the DNA of strain No. 1 performed by a standard method using a lytic solution based on 6M guanidinosuccinic preliminary disinfection of culture by adding thimerosal sodium concentrations up to 1:10000, followed by heating at a temperature of 56°C for 30 minutes (the Organization of work in research by PCR material infected with microorganisms of I-II pathogenicity groups. MU 1.3.1794-03).

Polymerase chain reaction is carried out in a volume of 25 μl; the reaction mixture contains: 1x buffer (10x Ptrbuffer - 2,5 mm), MgCl22.0 mm dNTP mixture - 0,3 mm oligonucleotide primers - 2 pmol, Taq DNA polymerase, and 0.1 units studied DNA 10 ál. Amplification products analyzed in 3% agarose gel with the addition of ethidium bromide and viewed under UV light.

Determine the sizes of the resulting fragments terC genes, ilvN and inv. They are 300, 515, 877 BP, which corresponds to the size of fragments of genes in strains of the main subspecies. The investigated strain No. 1 referred to the main subspecies of Y.pestis.

Example 2. Differentiation of strain No. 2 carried out analogously to example No. 1.

Dimensions formed in PCR fragments of genes terC, ilvN and inv amount 389, 560 and 877 BP, which corresponds to the sizes of the fragments produced in strains of the plague pathogen in non-core races. Strain No. 2 referred to as non-core subspecies of Y.pestis.

Example 3. Differentiation of strain No. 3 carried out analogously to example No. 1.

Dimensions formed in PCR fragments of genes terC, ilvN and inv amount 389, 560 and 169 BP, which corresponds to the sizes of the fragments produced in strains of the causative agent of pseudotuberculosis. Strain No. 3 referred to Y.pseudotuberculosis.

Thus, the claimed method of differentiation of strains of the plague pathogen main and non-main subspecies PCR, based on the variability of gene sequences livelihoods terC, ilvN and inv allows you to quickly, efficiently and reliably carry out the differentiation of States the MOU Y.pestis main and non-main subspecies and Y.pseudotuberculosis.

Table
Species, subspeciesDimensions genes
terCilvNinv
Y.pestis main subspecies300515877
Y.pestis non-main subspecies389560877
Y.pseudotuberculosis389560169

The method of differentiation of strains of the plague pathogen main and non-main subspecies and the causative agent of pseudotuberculosis, including DNA isolation, PCR with amplification of gene fragments of the studied strain, characterized in that when carrying out PCR using nucleotide primers for the genes terC, ilvN and inv with the following sequence:
89-S - AATCAAATCTCGCCCAGC,
89-As - GCTGCGTATCATTTCACC;
45-S - AGTGGTCTGCTTCTCTGG,
45-As - CGGCATACACAGAATACC;
inv839 - TACCTGCACTCCCACAAC,
invl007 - CCCATACGCTGATCTACC;
differentiation of strains is carried out by comparison of the sizes of the obtained gene fragments terC, ilvN and inv issleduemymi with similar fragments in the typical strains of plague primary, non-main subspecies and Yersinia pseudotuberculosis microbe, with typical strains of the plague pathogen main subspecies are formed in PCR fragments of genes terC - 300 gel, ilvN - 515 P.N. and inv - 877 P.N.; minor strains of the subspecies, respectively, 389, 560 and 877; and the strains of Yersinia pseudotuberculosis microbe - 389, 560 and 169.



 

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