RU2332464C1 - Method of plague and pseudotuberculosis microbe differentiation with parallel interspecies differentiation of plague microbe strains - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: invention concerns medical microbiology. The method involves extraction of the analysed strain DNA, polymerase chain reaction (PCR) using synthetic TAN1 and TAN2 primers complementary to hutG - YP01967 zone and featuring the following nucleotide sequences: TAN1-5'-TGGGCTTGAATACGGATGATG-3', TAN2-5'-ACAACCATGCTGACGTGGG-3'. Species and subspecies of the analysed microbe strains are defined by comparing amplicone volume produced by the analysed strains with markers, particularly with a mix of amplicones produced with TAN1 and TAN2 primers, and reference plague and pseudotuberculosis microbe strain DNA.

EFFECT: fast and accurate differentiation of plague and pseudotuberculosis microbe strains and inter-species differentiation of plague microbe strains.

3 cl, 3 dwg, 6 ex

Description

The invention relates to the field of medical microbiology, in particular to the differentiation of plague and pseudotuberculosis pathogens and molecular genetic typing of plague microbe strains of different origin.

The differentiation of two closely related and highly homologous species of the Yersinia genus - the plague microbe (pathogenicity group I) and the much less dangerous pseudotuberculosis pathogen (pathogenicity group III) is important in monitoring natural plague foci. Timely identification of activation of the natural focus allows using anti-epidemic measures to prevent the occurrence of diseases among people living or working in the zone of the active natural focus of plague. At the same time, elucidation of the origin of the plague microbe strain is of great importance for determining its potential danger. The strains of the main subspecies are classified as the most virulent and epidemiologically significant, strains of non-main subspecies (Altai, Hissar, Caucasian, Udege) and the group of Talas strains are considered to be of little epidemiological significance with selective virulence. Of particular importance is the ability to quickly identify and determine the origin of the strain in terrorist acts using pathogenic biological agents and detect imported cases of this especially dangerous infection.

Various methods of molecular genetic typing are used to differentiate strains of the plague microbe, such as IS typing, ribotyping, pulse electrophoresis, multilocus sequencing, VNTR typing. However, molecular typing methods based on DNA or RNA probing, macrorestriction analysis with pulsed field electrophoresis, sequencing are laborious, require the use of a radioactive or non-radioactive label and the use of expensive equipment (a vacuum apparatus for transferring DNA fragments from gel to membranes, an apparatus for hybridization, apparatus for electrophoresis in a pulsed field, sequencer).

Widespread methods for the indication, identification and differentiation of infectious pathogens based on PCR technology. The advantages of these methods are ease of execution, quick results, relatively inexpensive equipment and supplies.

To indicate the causative agent of the plague in methods based on the polymerase chain reaction, individual chromosomal or plasmid genes are used as the target DNA, most often pla, caf. lcrV or a combination of two or three genes (Hinnebusch BJ, 1998; Leal NC, Almedia AM, 1999; Neubauer H. et al., 2000; Rahalison L. et al., 2000; Hongwei ZJL, 2000; Balakhonov S.V. et al. 1999, 2000; Garanina, S. B., 2001, I. Suchkov et al., 2001). These methods make it possible to identify the plague microbe in any biological material without isolating a pure culture and in some cases give an idea of the potential virulence of the strain. However, for typing, determining subspecies affiliation, they are not suitable.

Closest to the proposed invention in terms of essential features is a method for differentiating avirulent due to loss of pigmentation region and virulent plague microbe strains (RU 2288275 C1, 11.27.2006), including isolation of total DNA, PCR with primers IH 1-5'TTT-ACC- GCA-ACA-ACA-TCA-TCC-3 ', IY 4-5'GGG-CTG-AAA-CCA-CTG-AGA-TG-3', identification and subsequent analysis of specific amplicons. However, in this case, a narrow problem is solved - the differentiation of virulent and avirulent strains of the plague microbe. The method does not allow for subspecies differentiation and differentiation with closely related strains of the pseudotuberculosis microbe.

The technical result of the invention is the ability to quickly, easily execute and interpret the results of differentiation of strains of plague and pseudotuberculosis microbes and simultaneous intraspecific differentiation of strains of plague microbe.

The essence of the invention lies in the fact that total DNA is isolated from the test material, carried out under certain conditions and PCR amplification mode with TAN1 and TAN2 primers complementary to the hutG - YP01967 region, amplicons of a certain size specific for pseudotuberculosis are detected by agarose or polyacrylamide gel electrophoresis and each of the subspecies of the plague microbe, with the primers having the following nucleotide sequences:

TAN1-5'-TGGGCTTGAATACGGATGATG-3 '

TAN2-5'-ACAACCATGCTGACGTGGG-3 ',

differentiation (species and intraspecific) is carried out by comparing the amplicons of the studied strains with markers. As markers, it is possible to use standard markers or a mixture of amplicons formed with TAN1-TAN2 primers and DNA of reference strains of plague and pseudotuberculosis microbes. As a standard marker, it is possible to use pUC 19 DNA treated with MspI restrictase.

The following strains are used as reference strains of the plague microbe:

- for the main subspecies — strains 231 from the Aksay highland center, A 1822 from the Kyzylkum desert center, A 1270 from the Transbaikal steppe center, 805 from the Priaraksinsky low mountain center;

- for the Udege subspecies - strains from Mongolia: I3069 from the aimak Uburkhangai and I2422 from the Bayan-Ulgeey aimag.

- for the Altai subspecies - strains of the Altai hotbed: I2183 from Ulandrik and I2359 from the Kosh-Agach region;

- for the Hissar subspecies - strain A 1249 from the Hissar highland focus;

- for the Talas group, strain A1802 from the Talas high mountain center;

- for the Caucasian subspecies - strain 1146 from the Zangezuro-Karabakh mountain center.

As reference strains of the pseudotuberculosis microbe, strains I of serotype A2526 (Alma-Ata region) and MollaretI are used.

The differentiation of plague and pseudotuberculosis microbes, as well as the determination of subspecies belonging to the size of the formed amplicons. To accurately determine the size of the amplicons formed from the DNA of the reference strains and used as a marker, they were sequenced.

During sequencing, it was found that strains of the pseudotuberculosis microbe of I serotype isolated in the Alma-Ata region and in France form amplicons of 692 bp in size. and 711 bp respectively. The strains of the plague microbe of the main subspecies are divided into three groups according to the size of the formed amplicons: the first group is represented by a strain from the Aksai high-mountainous outbreak - the amplicon size is 364 bp, the second group is represented by strains from the Transbaikal steppe. Tuva mountain foci and Mongolia - the size of the amplicon is 356 bp, the third group is represented by strains from the Kyzylkum desert, Priaraksinsky low mountain foci - the size of the amplicon is 348 bp. The strains of the Ulege subspecies are divided into two groups according to the size of amplicons: the first group is represented by strains from Mongolia, aimak Uburkhangay - amplicon size 416 bp, the second group - strains from Mongolia, Bayan-Ulgey aimag - amplicon size 348 bp The third group of the main subspecies and the second group of the Ulgean subspecies have amplicons of the same size, but differ in nucleotide composition. Upon detection of amplicons of 348 bp in size amplicon sequencing is recommended to differentiate strains from the indicated foci of the main and Udege subspecies. The strains of the Altai subspecies are divided into two groups: the first group is represented by a strain from the Altai mountain center, the Kosh-Agach region - the amplicon size is 396 bp, the second group is represented by strains from the Altai mountain center, from Ulandrik - the size of the amplicon is 405 bp. The strains of the Hissar subspecies circulate only in the Gissar highland focus and form amplicons of 456 bp in size. Strains of the Talas group circulate only in the Talas high mountain center and form amplicons of 380 bp in size. The strains of the Caucasian subspecies from the Zangezuro-Karabakh mountain center form amplicons 1819 bp in size, from the Prissevan mountain center 1807 bp, from the Priaraksinsky low-mountain center 1831 bp, from the Leninakan mountain and Tersko-Sunzhensky low-mountain center - 1803 p .n. Differences of 10-30 bp in fragments of this magnitude are not detected upon separation by gel electrophoresis. In this regard, only one strain from the Zangezuro-Karabakh mountain center is used as a reference strain of the Caucasian subspecies. Data allowing to correlate the size of the amplicons of the studied strains with a certain type, subspecies of microorganisms and the place of its persistence are presented in the table (see Fig. 3).

The method is as follows.

Isolation of total DNA from the test material suspected of being infected with a plague or pseudotuberculosis microbe is carried out according to the standard method for Y. pestis. (Organization of work during PCR studies of a material infected with microorganisms of pathogenicity groups I-II (MU 1.3.1794-03). The polymerase chain reaction is carried out according to a standard method using a pair of primers TAN1 - 5'-TGGGCTTGAATACGGATGATG-3 'and TAN2-5' -ACAACCATGCTGACGTGGG-3 ', complementary to the hutG — YP01967 region. Primers were annealed at 63 ° C. for 30 s with an amplification cycle of 35.

PCR products are analyzed on a 3.5% agarose or 5% polyacrylamide gel using TBE buffer according to a standard technique. The species, subspecies and focal affiliation of the microorganism is determined by comparing the size of the formed amplicon of the studied strains with markers.

A distinctive feature of the proposed method is the use of one pair of primers TAN1 and TAN2, which allows to simultaneously determine the species and subspecies of the studied strains of pathogenic Yersinia.

The claimed method has been successfully tested on seventy-two strains of different subspecies, including type strains Y. pestis 231 (708), A 1822 - main, Y. pestis 1146 - Caucasian, Y. pestis A-1249 - Hissar, Y.pestis KM683 (I2359) - Altai, Y. pestis I-3069, I2422 Ulegei subspecies, Y. pestis A-1802 (21/10) - the Talas group and sixty-eight strains of the pseudotuberculosis microbe.

The invention is illustrated by electrophoregrams of amplicons of the studied strains with standard markers and markers based on amplicons of reference strains.

The analysis results are presented in FIG. 1 and 2.

In FIG. Figure 1 shows the electrophoregram of amplicons obtained by PCR analysis of reference strains of plague and pseudotuberculosis microbes using TAN1 and TAN2 primers, which shows the position of the amplicons relative to the standard marker in 5% polyacrylamide gel, which characterizes the species, subspecies, and focal accessory.

1. Standard pUC 19 DNA marker treated with MspI restrictase

2. Y.pestis 231 - the main subspecies (Aksai mountain center)

3. Y.pestis A 1822 - the main subspecies (Kyzylkum desert center)

4. Y.pestis A1270 main subspecies (Transbaikal steppe outbreak)

5. Y. pestis 805 main subspecies (Priaraksinsky low mountain center)

6. Y.pestis A1249 - Hissar subspecies (Gissar highland)

7. Y.pestis A1802 - Talas group (Talas highland hearth)

8. Y.pestis I2359 - Altai subspecies (Altai highland focus)

9. Y.pestis I3069 - Udege subspecies (Mongolia, aimak Uburkhangai)

10. Y.pestis I2422, Udege subspecies (Mongolia, Bayan-Ulgei aimak)

11. Y.pestis I2183 - Altai subspecies (Altai high mountain center)

12. Y.pestis 1146 - Caucasian subspecies (Zangezuro-Karabakh outbreak)

13. Y.pseudotuberculosis A2526 (Alma-Ata) (I serotype)

14. Y. pseudotuberculosis Mollaret I (I serotype)

In FIG. Figure 2 shows the electrophoregram of amplicons obtained by PCR analysis of strains of plague and pseudotuberculosis microbes of different origin using primers TAN1 and TAN2, which shows the position of amplicons relative to the marker from amplicons of reference strains of plague and pseudotuberculosis microbe in 3.5% agarose gel, characterizing a species of 3.5% agarose gel and focal accessories.

16. A marker from amplicons of reference strains of the plague and pseudotuberculosis microbe

2. Y.pestis 231 - the main subspecies (Aksai mountain center)

17. Y.pestis A1824 - main subspecies (Kyzylkum desert center)

18 Y.pestis A1252 - main subspecies (Transbaikal steppe outbreak)

19. Y.pestis 813 - main subspecies (Priaraksinsky low-mountain center)

20. Y. pestis A 1723 - Hissar subspecies (Gissar highland)

21. Y.pestis A1814 - Talas group (Talas highland hearth)

8. Y.pestis I2359 - Altai subspecies (Altai highland focus)

22. Y.pestis I3086 - Udege subspecies (Mongolia, aimak Uburkhangai)

23. Y.pestis I3068 - Ulgean subspecies (Mongolia, aimak Uburkhangai)

24. Y.pestis I3071 - Ulgean subspecies (Mongolia, aimak Hurmen Som)

25. Y.pestis I2817 - Altai subspecies (Altai highland focus)

26. Y. pestis 542 Az. - Caucasian subspecies (Zangezuro-Karabakh outbreak)

13. Y.pseudotuberculosis A2526 (Alma-Ata) (I serotype)

14. Y. pseudotuberculosis Mollaret I (I serotype)

Marker 16 contains amplicons obtained by amplification of DNA of reference strains of plague and pseudotuberculosis microbes with the proposed primers TAN1 and TAN2.

To obtain a marker, PCR is carried out with reference strains according to the scheme described in Example 1. Among the reference strains forming amplicons of the same size, one of the strains is selected. Then the reaction mixtures containing amplicons are combined and precipitated with DNA by adding ethanol and centrifugation. The precipitate is dried and dissolved in deionized water. The resulting solution contains a set of amplicons characteristic of all reference strains, and is used as a marker for differentiation of strains. When dried, marker amplicons can be stored at room temperature for a long time.

The presence of a DNA fragment of a certain size on the electrophoregram allows you to attribute the studied strain to a specific species and to determine the plague microbe to determine the subspecies affiliation.

The invention is illustrated by the following examples.

Example 1. Identification of the strain Y. pestis 231 (708) of the main subspecies from the Aksai mountain center. (Model experiment).

DNA preparation

In a model experiment, strain Y. pestis 231 (708) was plated on LB agar and grown overnight at 28 ° C. Total DNA is extracted from the test material containing the Y. pestis 231 (708) strain of the main subspecies.

According to the turbidity standard, a suspension of microorganisms is prepared in distilled water at a concentration of 1.0 × 10 ⁷ -1.0 × 10 ⁹ m.k. / ml (microbial cells in 1 ml).

Disinfection is carried out by adding sodium thiolate to a concentration of 1: 10000, followed by heating at 56 ° C for 30 minutes. After treatment with sodium thiomethylate, 100 μl of the sample was transferred to 1.5 ml microcentrifuge tubes and a lysing solution based on 6 M guanidine isothiocyanate was added in a volume of 300 μl and incubated at 65 ° C for 15 minutes. After this stage, the material is considered disinfected (Clause 1.1. MU 1.3.1794-03).

At the end of incubation and lowering the temperature of the sample to room temperature, 30 μl of the nucleosorbent are introduced into the test tube, shaken and incubated for 10 minutes, periodically mixing the sorbent to a homogeneous state 3-4 times.

The tube is centrifuged for 30 s on a microcentrifuge, the supernatant is removed.

300 μl of a solution based on 4 M guanidine isothiocyanate is added to the precipitate and the tube is shaken on a vortex, after which the sample is centrifuged for 30 s, the supernatant is removed.

1 ml of washing buffer is added to the precipitate, the tube is shaken until the suspension is homogeneous, centrifuged for 40 s and, as accurately as possible, the supernatant is removed using the tip of the dispenser.

The tubes are placed in a solid state thermostat at a temperature of 55-65 ° C for 15-20 minutes, leaving them open to evaporate the residual alcohol contained in the wash buffer. One should strive to dry the nucleosorbent as thoroughly as possible, since more complete desorption of DNA is associated with this and, in addition, the presence of alcohol can cause inhibition of PCR.

25-50 μl bidistilled water is added to test tubes with a dried sorbent, they are closed, vortexed until a sorbent is homogeneous and incubated for 8-10 minutes at a temperature of 55-65 ° C, shaking 2-3 times.

Samples are centrifuged for 1 minute. The supernatant is used as a test material for PCR. Prepared samples can be stored at 4 ° C for a week or at minus 20 ° C for up to 6 months.

Carrying out PCR.

The polymerase chain reaction is carried out in 0.5 ml tubes.

The reaction mixture in a volume of 25 μl contains 1 · PCR buffer (10 · PCR buffer - 2.5 μl), MgCl ₂ - 2.0 mM, dNTPmix - 0.3 mM, primer TAN1-2 pmol, primer TAN2 - 2 pmol, Taq DNA polymerase - 0.1 units, test DNA - 10 μl.

Amplification mode: initial denaturation of 94 ° C for 2 min, then 35 cycles are carried out: denaturation at 94 ° C for 30 s, annealing at 63 ° C for 30 s, elongation at 72 ° C for 40 s.

Amplification products are analyzed on a 3.5% agarose gel. 3 μl of the reaction mixture is applied to the gel; phage λ DNA treated with PstI restriction enzyme, pUC 19 DNA treated with MspI restriction enzyme is used as a marker.

The agarose gel is stained with ethidium bromide and viewed under ultraviolet radiation by comparing the resulting amplicon with a marker to determine the size of the fragment. The presence of a 364 bp fragment indicates the belonging of this strain to the main subspecies from the Aksai highland focus.

Example 2. The identification of the strain Y. pestis KM683 (I2359) - Altai subspecies from the Altai mountain center is carried out analogously to example 1.

In the analysis of amplification products, the length of the synthesized amplicon was 396 bp, which indicates that this strain belongs to the Altai subspecies from the Altai mountain center.

Example 3. The identification of strain Y. pestis A-1249 - Hissar subspecies is carried out analogously to example 1.

In the analysis of amplification products, the length of the synthesized amplicon was 456 bp, which indicates that this strain belongs to the Hissar subspecies from the Hissar highland focus.

Example 4. The identification of the strain Y. pestis I-3069 - Udege subspecies is carried out analogously to example 1.

In the analysis of amplification products, the length of the synthesized amplicon was 416 bp, which indicates that this strain belongs to the Ulgean subspecies from Aimak Uburkhangai from Mongolia.

Example 5. The identification of strain Y. pestis A-1802 (21/10) - Talas group is carried out analogously to example 1.

In the analysis of amplification products, the length of the synthesized amplicon was 380 bp, which indicates that this strain belongs to the Talas group from the Talas highland focus.

Example 6. The identification of strain Y. pestis 1146 - Caucasian subspecies is carried out analogously to example 1.

In the analysis of amplification products, the length of the synthesized amplicon was 1819 bp, which indicates that this strain belongs to the Caucasian subspecies from the Zangezuro-Karabakh outbreak.

Example 7. The identification of the strain Y. pseudotuberculosis A-2526 is carried out analogously to example 1.

When analyzing amplification products, the length of the synthesized amplicon was 692 bp, which indicates that this strain belongs to the species Y. pseudotuberculosis (serotype I) isolated in the Almaty region.

The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information and identification of sources containing information about analogues of the invention, made it possible to establish that no sources were found that are characterized by signs that are identical to all the essential features of the invention.

Thus, the claimed method, based on a polymerase chain reaction using a selected pair of primers, allows you to quickly identify plague and pseudotuberculosis microbe strains with the same subspecies differentiation of plague microbe strains in compliance with the rules for working with especially dangerous infections, which makes it possible to use this method in research institutions, anti-plague stations and in the work of Specialized Anti-Epidemic Brigades.

Claims (3)

1. A method for differentiating plague and pseudotuberculosis microbes with simultaneous intraspecific differentiation of plague microbe strains, including isolation of total DNA of the studied strain, polymerase chain reaction using synthesized primers, followed by analysis of specific amplicons, characterized in that TAN1 primers are used for setting up the polymerase chain reaction TAN2 complementary to the hutG-YP01967 region of the following nucleotide sequence: TAN1-5'-TGGGCTTGAATACGGATGATG-3 ' TAN2-5'-ACAACCATGCTGACGTGGG-3 ', differentiation of strains is carried out by comparing the amplicon size of the test strain with a marker. 2. The method according to claim 1, characterized in that standard markers are used as a marker. 3. The method according to claim 1, characterized in that as a marker a mixture of amplicons formed with TAN1-TAN2 primers and DNA of reference strains of plague and pseudotuberculosis microbes is used.

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