RU2360972C1 - Method detection and evaluation of biotype, serogroup and toxigenicity of cholera agent and related kit - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of detection and evaluation of biotype, serogroup and toxigenicity of cholera agent with using the multipoint polymerase chain reaction (PCR) technique includes: DNA separation from the analysed object, one stage PCR production with using three pairs of the synthesised primers to a site of wbeN gene of wbf cluster, coding 01-antigen synthesis, to a site of ctxA gene coding synthesis of subunit A of cholera enterotoxin and to a site specific for comma bacillus of biovar eltor hlyA gene coding hemolysin synthesis; annealing of primers at temperature 95°-60°C during 13.5 min at number of amplification cycles 35; gel-electrophoresis; analysis of PCR results from electrophoregram and estimated results of detection and evaluation of required characteristics of comma bacillus from the presence or absence of specific strips of amplified DNA. A kit used in relation to the method comprises a complete set of reagents for DNA separation from a material, a complete set for amplification containing the primers specific to wbeN gene, hlyA gene and ctxA gene of comma bacillus, positive and negative control samples, a complete set for analysis of the products.

EFFECT: method with using the kit provides rapid, simple and precise detection of cholera agent and evaluation of its three basic properties.

3 cl, 5 dwg, 3 tbl

Description

The invention relates to medicine, in particular to medical diagnostics, specifically to a method and kit for detecting DNA of a cholera pathogen in material from sick people and from environmental objects, and can be used in research institutions, regional and regional sanitary and epidemiological surveillance services.

The laboratory diagnosis of cholera is based on cultural research methods. However, when determining the serovar and biovar, the isolated strains of Vibrio cholerae encounter difficulties associated with their phagoresistance to cholera diagnostic phages (which determine the classical and eltor biovars), as well as their reduced agglutination by cholera diagnostic sera (which determine serovar 01 or not 01). Polymerase chain reaction (PCR) is used as an accelerated method for the detection of the pathogen, as well as at the stage of determining the epidemic significance (toxigenicity) of selected cultures.

Currently, one of the most promising methods for the diagnosis of viral and bacterial diseases of humans and the indication of their pathogens, in particular the indication of Vibrio cholerae, is widely used PCR analysis based on the detection of pathogen DNA.

A known method for detecting a cholera pathogen, including DNA isolation, PCR, consisting of primary denaturation, a cycle from denaturation of annealing and synthesis, final synthesis, as well as an appropriate diagnostic kit. The kit contains reagents for PCR, water, dNTP (deoxynucleotide triphosphates) enzyme, control DNA, mineral oil and ctx2, ctx3 primers providing amplification of the ctx A fragment of the gene encoding the synthesis of the A subunit of cholera toxin, 564 bp in size. (“GenKhol - Test system for detecting V. cholerae ctxA + DNA by polymerase chain reaction”, TU 8895-006-01898109-2007).

The disadvantage of this method and kit is that with their help it is impossible to carry out multilocus analysis of several genes simultaneously, which slows down the process of researching the material. In addition, the described method and kit does not allow to detect strains of cholera vibrios that do not contain the ctx A gene, but which may have other virulence factors.

A known kit for determining DNA of periodontopathogenic microbes Prevotella intermedia sensu stricto, Bacteroides forsythus, Treponema denticola, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis using multilocus polymerase chain reaction. The kit includes a sample reagent kit, an amplification kit containing primers specific for Porphyromonas gingivalis DNA, Actinobacillus actinomycetemcomitans, Treponema denticola, Bacteroides forsythus, Prevotella intermedia sensu stricto, positive and negative control samples, and a PC product analysis kit. The kit provides simultaneous, quick, simple and accurate detection of 5 types of periodontopathogens in one sample (Patent RU 2306341, 09/20/07). However, the described kit is intended to detect microorganisms not belonging to the Vibrionocae family and does not provide for the detection of cholera vibrio.

Closest to the proposed invention, the technical solution is a method for determining the toxigenicity and biovar of cholera vibrios using a set of primers that simultaneously amplify two fragments of the ctx A2-B gene encoding the synthesis of V. cholerae cholera toxin and three fragments of the hly A gene encoding hemolysin synthesis. For amplification of two fragments of the ctx A2-B gene, two pairs of primers C2F-C2R and C4F-C4R are used, and for the hly A gene, three primers are used - H4F, H3R and H3F. The described primers (C2F, C2R, C4F, C4R, H4F, H3R and H3F) in one reaction mixture provide simultaneous amplification of fragments of the ctxA 2-B and hly A genes in a two-stage reaction.

ctx A2-B

C2F - 5'-AGGTGTAAAATTCCTTGACGA-3 ',

C2R - 5'-TCCTCAGGGTATCCTTCATC-3 ',

C4F - 5'-ACTGATTTGTGTGCAGAATACCAC-3 ',

C4R - 5'-TGATAGCCATCTCTCTTTTTTCCAG-3 '.

hly a

H4F - 5'-GCAAACAGCGAAACFAATACC-3 ',

H3R - 5'-TCCACCCCACCAGTCACC-3 ',

H3F - 5'-GGGAGCCGGCATTCATCT-3 '.

When implementing the method, an amplification program is used, including preliminary denaturation at 95 ° C for 2 min, 35 cycles (95 ° C for 60 s, 60 ° C for 90 s, 72 ° C for 90 s), final elongation for 10 min at 72 ° C. The considered method for determining the ctxA and hlyA genes involves performing studies in two stages, which takes up to 5 hours.

The disadvantage of this method and kit is that the primer mixture described above for the detection of two hlyA and ctx A-B genes does not allow the simultaneous determination of the pathogens serogroup, which is very important when indicating the cholera pathogen and assessing its epidemic significance. In addition, the amplification program used is not effective at the simultaneous detection of three hlyA, ctxA, and wbeN genes, which determines the pathogens serogroup, and a two-stage formulation of the reaction increases the risk of contamination of samples with amplicons when transferring PCR - the product of the first stage to the reaction mixture for the second stage, complicates the formulation reaction and increases the time spent on obtaining results.

The objective of the invention is the creation of a highly specific, sensitive, simple and fast-performing method and an appropriate kit for the detection and identification of cholera vibrio in the material from sick people and environmental objects using primers that simultaneously identify and evaluate the causative agent of cholera - Vibrio cholerae according to three criteria : serogroup, biovar, toxigenicity (epidemic significance).

The technical result achieved by the claimed group of inventions is highly specific and rapid identification of the causative agent of cholera with the simultaneous determination of biovar, serogroup, toxigenicity.

The technical result is achieved by a method for indicating and identifying the causative agent of cholera - Vibrio cholerae in the material from patients and from environmental objects, in which three pairs of primers were selected and synthesized for one-stage PCR:

to the wbeN site of the wbf gene cluster encoding the synthesis of 01 - antigen,

- wbeN1 - 5'-ATCAGTTTGCTTGCGCTTCTCC-3 ',

- wbeN2 - 5'-ATCTGGACATCGCATCGCAC-3 ',

providing the formation of a fragment of 420 bp;

to the ctx A region of the gene encoding the synthesis of the A subunit of cholera enterotoxin,

- ctx2 - 5'-GGAGCCGGCATTCATCTGAA-3 ',

- ctx3 - 5'-TTTCGGGCCATCTCCACTGA-3 ',

providing the formation of a fragment of 564 bp;

to the site of a specific for cholera vibrios biovar eltor hly A gene encoding the synthesis of hemolysin,

hly1 - 5'-CGGGCAGATTCTAGACCTG-3 ',

hly2 - 5'-CGATGATCTTGGAGCATTCCCAC-3 ',

providing the formation of a fragment measuring 264 bp;

amplified according to the program for amplifiers with temperature control according to the matrix: preliminary denaturation of 95 ° С - 5 min, 5 cycles from 95 ° С - 40 s, 62 ° С - 40 s, 72 ° С - 40 s, 30 cycles from 95 ° С - 30 s, 60 ° С - 30 s, 72 ° С - 30 s, final completion of the chain 72 ° С - 5 min, and for applicators with active temperature control (according to the solution in vitro): 95 ° С - 5 min, 5 cycles from 95 ° C - 10 s, 62 ° C - 10 s, 72 ° C - 10 s, 30 cycles from 95 ° C - 7 s, 60 ° C - 7 s, 72 ° C - 7 s, final completion of the chain 72 ° C - 5 min, taking into account the results of the reaction by the presence or absence of a specific electrophoregram amplified DNA bands when compared with control samples.

The technical result is also achieved by a kit for the detection and identification of the cholera pathogen, including components for PCR: 2-fold buffer solution with bromophenol blue and ficoll 400, pH 8.5, mineral oil, deionized sterile water, positive control, Taq polymerase enzyme, a mixture of four deoxynucleotide triphosphates, a mixture of oligonucleotide primers WbeN1 - WbeN2, ctx2-ctx3, hly1-hly2 in a ratio of 1.5: 1.2: 1, respectively, the components are separated by a 20% paraffin layer into two reaction mixtures, one of which contains 5 μl dNTF solution and primers: wbeN1 5'-ATCAGTTTGCTTGCGCTTCTCC-3 'and wbeN2 5'-ATCTGGACATCGCATCGCAC-3', hly1 5'-CGGGCAGATTC-3GTG-5GCTAT-CTGATCTAGACCTG-3 ' 'and ctx3 5'-TTTCGGGCCATCTCCACTGA-3', the second mixture is a 2-fold buffer into which the Taq polymerase enzyme located in a separate microtube is introduced ex tempore, as well as components for DNA isolation and analysis of diagnostic results.

The synthesis of oligonucleotides is carried out by the phosphoamitide method. For example, on an ASM-800 DNA synthesizer from Bioset.

Justification for the selection of primers.

The possibility of biotyping the toxigenic strains of cholera vibrios by amplification in PCR of the top A gene, the nucleotide sequence of which is different in the classical and Eltor biovars (Keasler SP, Hall RH / Lancet. - 1993. - Vol.341. - P.1661; Karaolis DKR et al . PNAS. - 1998. - Vol. 95. - P.3134-3139). However, V. cholerae strains that do not contain ctx A and topA genes are often isolated from vibriocarriers and from environmental objects. We selected the hly A gene, which is present in the genome of all cholera vibrios, but the classical biovar contains a deletion of 11 bp in size.

To identify the O1 serogroup of Vibrio cholerae, primers were selected that amplified the 420 bp wbeN gene region. According to S. Yamasaki et al. [FEMS Microbiol. Letters. - 1999 .-- Vol. 179. - P.115-121] said fragment is included in the group of gmd-wbeO genes that are found only in cholera vibrios 01 of the serogroup.

Based on the nucleotide sequences of the selected genes presented in the NCBI GenBank database, using the Primer Premier-V5 program (Premier Bio Soft International) and the BLAST algorithm, the wbeN1 - wbeN2 oligonucleotide primers for the wbeN gene included in the cluster of genes responsible for the synthesis of 01 antigen were selected as well as hly1-hly2 primers on the hly A gene, the nucleotide sequence of which is specific for cholera vibrios of the biovar eltor and cholera vibrios of the non-01 serogroup.

Known primers complementary to the ctxA gene encoding the cholera toxin A subunit were used to determine the epidemic significance of Vibrio cholerae strains [Fields P.I. et al. J. Clin. Microbiol. - 1992. - P.2128-2121].

The specificity of the primers was evaluated by multiple sequence comparison using software such as BLAST (Lastaew to evaluate polymorphisms in the compared sequences).

Primers wbeN1 - wbeN2 are located on the site of the wbeN gene and initiates amplification of the product 420 bp (figure 1).

Hly1-hly2 primers are located on the hlyA gene region and initiate amplification of the product 264 bp (figure 2).

These primers do not form secondary structures (hairpins) and dimers, it contributes to their effective binding to matrix DNA.

The optimal composition of the reaction mixture for performing multilocus PCR was experimentally established. The selected concentration of primers wbeN1-wbeN2, ctx2-ctx3, hly1-hly2 is in the ratio of 1.5: 1.2: 1, respectively, of the Tag enzyme - polymerase at a concentration of 1.25 units. necessary and sufficient to ensure high specificity and efficiency of the reaction and allow to obtain easily detectable amounts of amplicons.

When using the temperature control mode for the matrix and for the solution in the test tube, the temperature range is determined, the primer annealing time, and the effective number of cycles (table 1).

Analytical characteristics.

A preliminary assessment of the analytical specificity of the primers used using the BLAST on-line server (blast) showed the lack of homology of the selected sequence with other types of microorganisms at a significance level of E = 10.

The sensitivity and specificity of multilocus PCR was experimentally evaluated by 10-fold dilution of the following cultures: 5 V. cholerae classica strains, 10 V. cholerae eltor ctx A ⁺ , 10 V. cholerae eltor ctx A ^- 10, V. cholerae O139 ctx A ⁺ , 10 - V.cholerae O139 ctx A ^- , 50 - V.cholerae non-01 non-O139, 7 - Escherichia coli, 6 - Shigella spp., 5 - Salmonella spp., 4 - Pseudomonas spp., 3 - Aeromonas spp., 3 - Alcaligenes faecalis, 3 - Comamonas spp., 3 - Corynebacterium diphteriae, 3 - Listeria monocitogenes, 3 - Plesiomonas shigelloides, 3 - Proteus vulgaris, 3 - Serracia marcescens, 2 - Enterococcus sroberund, 1 - , 1 - Erysepelotrix rhusiopthiae, 1 - Lactobacillus casei, 1 - Sarcina aurantica (table 2).

Figure 3 presents the specificity results of multiplex PCR:

1. V. cholerae classica 569 V - 1 × 10 ⁵ m.k. / ml,

2. V. cholerae classica 16126 - 1 × 10 ⁴ m.k. / ml,

3. V. cholerae eltor P15243 (ctxA +) - 1 × 10 ⁴ m.k. / ml,

4. V. cholerae eltor M-1344 (ctxA +) - 1 × 10 ⁴ m.k. / ml,

5. V. cholerae eltor 1 (ctxA +) - 1 × 10 ⁴ m.k. / ml,

6. E.coli 0111-BH-3133 - 1 × 10 ⁷ m.k. / ml,

7. V. cholerae eltor M-1231 (ctxA-) - 1 × 10 ⁶ m.k. / ml,

8. V. cholerae eltor M-1246 (ctxA-) - 1 × 10 ⁶ m.k. / ml,

9. V. cholerae non-O1 P-9741 - 1 × 10 ⁵ m.k. / ml,

10. V. cholerae eltor KM-26 (ctxA-) - 1 × 10 ⁵ m.k. / ml,

11. V.cholerae O28 12630-62 (ctxA-) - 1 × 10 ⁶ m.k. / ml ,,

12. K + - DNA of V. cholerae eltor M-1289 at a concentration of 1 ng / μl,

13. Negative control.

Figure 4 presents the sensitivity results of multiplex PCR:

with three pairs of primers for three target DNAs: ctxA gene (564 bp fragment), wbeN (420 bp fragment), hlyA (264 bp fragment) where:

1. K + - DNA of V. cholerae eltor M-1289 at a concentration of 1 ng / μl,

2. V. cholerae eltor M-1289 - 1 × 10 ⁵ m.k. / ml,

3. V. cholerae eltor M-1289 (ctxA +) - 1 × 10 ⁴ m.k. / ml,

4. V. cholerae eltor M-1289 (ctxA +) - 1 × 10 ³ m.k. / ml,

5. Negative control.

With two pairs of primers on wbeN and hlyA genes - to determine the O1 serogroup and biotyping:

6. K + - DNA of V. cholerae eltor M-128 9 at a concentration of 1 ng / μl,

7. V. cholerae eltor M-1289 (ctxA +) - 1 × 10 ⁵ m.k. / ml,

8. V. cholerae eltor M-1289 (ctxA +) - 1 × 10 ⁴ m.k. / ml,

9. V. cholerae eltor M-1289 (ctxA +) - 1 × 10 ³ m.k. / ml,

10. V. cholerae non-O1 P-9741 - 1 × 10 ⁴ m.k. / ml,

11. V. cholerae non-O1 P-9741 - 1 × 10 ³ m.k. / ml,

12. Negative control.

The concentration of microbial cells in bacterial suspensions according to control seeding was 0.7 × 10 ⁿ m.k./ml for V. cholerae eltor M-1289, 0.13 × 10 ⁿ m for V. cholerae non-O1 P-9741 .k./ml.

As a result of the assessment, it was found that the selected primers for detection and characteristics of cholera vibrios

wbeN1 -5'-ATCAGTTTGCTTGCGCTTCTCC-3 ',

wbeN2 - 5'-ATCTGGACATCGCATCGCAC-3 ',

hly1 - 5'-CGGGCAGATTCTAGACCTG-3 ',

hly2 - 5'-CGATGATCTTGGAGCATTCCCAC-3 ',

ctx2 - 5'-GGAGCCGGCATTCATCTGAA-3 ',

ctx3 - 5'-TTTCGGGCCATCTCCACTGA-3 '

possess high sensitivity - 1 × 10 ³ m.k / ml and specificity - 100%.

The inventive set of components for detection and characterization of the causative agent of cholera by multilocus PCR method is divided into 3 sets:

kit No. 1 contains components for DNA isolation, packaged in six plastic bottles containing solution 1 (6M guanidine thiocyanate solution), solution 2 (4M guanidine thiocyanate solution), solution 3 (alcohol-salt solution), acetone, eluent for DNA (TE buffer), 2 plastic test tubes with nucleosorbent;

kit No. 2 contains components for PCR, including 100 plastic tubes with reaction mixture 1 (primers and nucleotides under a paraffin layer), 1 plastic tube with reaction mixture 2 (buffer and deionized water), 1 plastic tube containing Taq polymerase, 3 plastic tubes with mineral oil, 1 plastic tube with TE buffer, 1 plastic tube containing a positive control sample;

set No. 3 contains components for analyzing the results of the analysis and includes 1 plastic bottle containing 50xTAE with ethidium bromide and 2 bottles with agarose for electrophoresis.

The proposed set of components is equipped with 10 plastic bottles and 109 plastic test tubes.

The kit is designed to detect DNA of the causative agent of cholera (Vibrio cholerae) in samples of biological material and environmental objects, to determine its serogroup, biovar and epidemic significance by the multilocus polymerase chain reaction. The kit is designed for 100 determinations, including control samples.

The inventive method for the detection of the pathogen of cholera includes the following steps.

a) Isolation of DNA (set No. 1).

c) PCR (kit No. 2).

PCR is carried out in one step using the principle of "hot start", reaching it by separation of the two reaction mixtures with a layer of 20% paraffin according to the following program for amplifiers with temperature control by matrix: preliminary denaturation of 95 ° C - 5 min, 5 cycles of 95 ° C - 40 s, 62 ° C - 40 s, 72 ° C - 40 s, 30 cycles of 95 ° C - 30 s, 60 ° C - 30 s, 72 ° C - 30 s, final chain completion 72 ° C - 5 min , and for applicators with active temperature control (according to the solution in vitro): 95 ° С - 5 min, 5 cycles from 95 ° С - 10 s, 62 ° С - 10 s, 72 ° С - 10 s, 30 cycles from 95 ° C - 7 s, 60 ° C - 7 s, 72 ° C - 7 s, final completion of the chain 72 ° C - 5 min, which significantly reduces the study time.

c) Analysis of the results (set 3).

The amplified DNA after PCR is detected by horizontal gel electrophoresis in 2% agarose. The results of PCR analysis are taken into account by the presence or absence of specific bands of amplified DNA on the electrophoregram when compared with control samples (positive and negative). The DNA of the Vibrio cholerae strain containing the hlyA, cthA, and wbeN genes was used as a positive control sample. An assessment of the data obtained is presented in table 3.

A method for detecting a cholera pathogen using the inventive kit is as follows.

1. Isolation of DNA is carried out using kit No. 1.

- The DNA isolation kit is removed from the refrigerator and kept at room temperature. All kit reagents are added with separate tips using automatic micropipettes.

- Solution 1 is heated at a temperature of 60-65 ° C until the crystals are completely dissolved, after which they are added to the disinfected samples in a volume of 300 μl. Samples are thoroughly mixed in a microcentrifuge / shaker and incubated at a temperature of 65 ° C.

- The sorbent is carefully resuspended in a microcentrifuge / shaker. 25 μl of the prepared sorbent is added to each tube, mixed on a microcentrifuge / shaker for 30 s and left in a rack for 2 minutes. The procedure is repeated twice. Then the tubes are centrifuged at 12000 rpm for 30 s, the supernatant is removed.

- 300 μl of solution 2 is added to the pellet. The contents of the tube are mixed on a microcentrifuge / shaker until homogeneous, centrifuged at 12,000 rpm for 30 s, the supernatant is removed.

- 500 μl of solution 3 is added to the pellet. The contents of the tube are mixed on a microcentrifuge / shaker until homogeneous and centrifuged at 12,000 rpm for 30 s. The supernatant is removed, washing with solution 3 is repeated.

- 400 μl of acetone is added to the precipitate. The contents are mixed on a microcentrifuge / shaker, then centrifuged at 12,000 rpm for 30 s, the supernatant is removed.

- The precipitate is dried at a temperature of 65 ° C for 5-7 minutes.

- 50 μl of TE buffer is added to the precipitate and maintained at 65 ° C for 10 minutes, shaking 2-3 times on a microcentrifuge / shaker. At the end, the suspension is centrifuged at 1200 rpm for 1 min. The supernatant contains purified DNA.

2. Carrying out PCR amplification of DNA (set 2).

- Remove from the refrigerator the required number of microtubes with the reaction mixture 1, corresponding to the number of test samples, as well as for positive and negative control.

- In a separate 0.6 ml microtube, 10 μl of reaction mixture 2 and 0.25 μl of Taq polymerase are combined for each sample, the resulting mixture is mixed on a microcentrifuge / shaker and transferred to 10 μl in prepared microtubes with reaction mixture 1, not damaging a layer of paraffin.

- In the tubes in the reaction mixture 1 contribute 10 μl of DNA from the studied samples and controls without damaging the paraffin layer.

- The microtubes are transferred to a thermal cycler preheated to a temperature of 95 ° C, and amplification is carried out according to the appropriate program for each type of temperature adjustment.

The PCR time is 1 h 30 min.

3. Analysis of the diagnostic results (set 3).

- Detection of amplified DNA after PCR is carried out by horizontal gel electrophoresis in 2% agarose.

- To prepare 50 ml of a 2% agarose gel, 50 ml of 1 × TAE ethidium bromide buffer are added to 1 g of agarose. The mixture is placed in a heat-resistant flask and heated in a boiling water bath until the agarose is completely melted, then it is cooled to a temperature of 50 ° C and poured onto a prepared table for pouring the gel.

- The results of PCR analysis are taken into account by the presence or absence of specific bands of amplified DNA on the electrophoregram. Evaluation of the data obtained is presented in table 3.

The analysis time is 2.5 hours.

An example of a specific implementation.

The test material was 2 stool samples weighing 1 g: one sample was intact, and the second was contaminated with the biovar eltor cholera pathogen to a final concentration of 1 × 10 ³ m.k./g, and 2 samples of open water from various sources, each with a volume of 0 , 5 l.

9 ml of saline was added to the stool samples and mixed thoroughly. The resulting suspension was transferred to 15 ml centrifuge tubes and centrifuged for 2-3 minutes at 5000 rpm to remove large particles. The supernatant was centrifuged for 15 minutes at 10,000 rpm. The pellet was resuspended in 100 μl of saline.

Water samples were fractionally centrifuged: initially for 2-3 min at 5000 rpm, then the supernatant was centrifuged for 15 min at 10000 rpm. The precipitate was resuspended in 0.1 ml of physiological saline.

To the samples prepared in this way, 300 μl of solution 1 from kit 1 was added (the solution was heated in a water bath before the study began until crystals were completely dissolved). Samples were incubated for 10 min at 65 ° C, after which 25 μl of the sorbent from kit 1 was added. Thoroughly mixed on a microcentrifuge / shaker and incubated at room temperature for 10 min. Stirring of the sorbent was carried out every 2 min. After which the samples were centrifuged at 12000 rpm for 30 s, the supernatant was removed.

Then the precipitate was washed once with solution 2 from set 1, twice with solution 3 from set 1 and once with acetone from set 1. After the last washing with acetone, the supernatant was removed most thoroughly. The precipitate was dried at a temperature of 65 ° C for 5-7 minutes.

Then, 50 μl of TE buffer was added to the precipitate and kept at a temperature of 65 ° C for 10 min, shaking in a microcentrifuge / shaker 2-3 times. At the end, the suspension was centrifuged at 1200 rpm for 1 min. PCR was performed using the resulting solution of isolated and purified DNA preparation using kit 2.

For this, 6 microtubes with the prepared reaction mixture 1: 4 tubes for the test samples and 2 for positive and negative control were taken out of the refrigerator compartment.

60 μl of a solution of the reaction mixture 2 from kit 2 (at the rate of 10 μl per tube) and 1.5 μl of Taq polymerase (which was removed from the freezer) were added to a separate 0.6 ml microtube. The resulting mixture was mixed on a microcentrifuge / shaker and transferred 10 μl into prepared microtubes with reaction mixture 1 without damaging the paraffin layer.

Then, 10 μl of 10 μl of DNA from the studied samples, DNA of the positive control and 10 μl of TE buffer were added to the tubes without damaging the paraffin layer.

The microtubes were transferred to the RTS-100 “MJ Research” thermal cycler, preheated to a temperature of 95 ° C and amplified according to the appropriate program for amplifiers with temperature adjustment by matrix: preliminary denaturation of 95 ° C for 5 min, 5 cycles of 95 ° C to 40 s, 62 ° C - 40 s, 72 ° C - 40 s, 30 cycles of 95 ° C - 30 s, 60 ° C - 30 s, 72 ° C - 30 s, final chain completion 72 ° C - 5 min.

After termination of thermal cycling, the samples were applied to an agarose gel and electrophoresis was performed for 20 min at 200 V. The agarose gel was prepared on 1 × TAE with ethidium bromide, the electrophoresis chamber was filled with a similar buffer. After the end of the process, the gel was washed in running water and placed on a UV transilluminator. Accounting and documentation of the results was carried out on a gel-documenting system GelDoc (BioRad).

On the electrophoregram (figure 5) were recorded the following fragments.

1. Sample 1. Intact bowel movements - no fragments.

2. Sample 2. Feces artificially infected with the biovar eltor cholera pathogen - three fragments corresponding to the size of the positive control fragments: 564, 420 and 264 bp

3. Sample 3. Water of open reservoirs 1 - two fragments corresponding in size to fragments of positive control 420 and 264 bp

4. Sample 4. Water of open reservoirs 2 — one fragment corresponding in size to the fragment of positive control 264 bp

5. Test positive control - three fragments of 564, 420 and 264 bp

6. Sample of negative control - no fragments.

The interpretation of the results was carried out in accordance with identification table 3.

No fragments were detected in the negative control — reaction results can be taken into account.

In the positive control, three fragments of sizes 564, 420, and 264 bp are detected. - the results of the reaction can be taken into account.

Sample 1. Intact bowel movements - negative result - DNA of cholera vibrios was not detected.

Sample 2. Stool, artificially infected with the causative agent of cholera biovar eltor - a positive result - DNA of toxigenic (epidemically dangerous) cholera vibrios O1 of the serogroup of the Eltor biovar was detected.

Sample 3. Water of open reservoirs 1 - a positive result - DNA of non-toxigenic (epidemically non-hazardous) cholera vibrios O1 of the serogroup of the Eltor biogar biogar was detected.

Sample 4. Water of open reservoirs 2 - a positive result - DNA of non-toxigenic (epidemically non-hazardous) cholera vibrios of non-O1 serogroup was detected.

An advantage of the claimed group of the invention is that the reaction is carried out in one step, which facilitates the analysis, reduces the time to obtain results by half and reduces the risk of contamination of samples with amplicons, increases the information content of PCR analysis. In addition, modifying the composition of the buffer reduces labor costs at the stage of recording results. Selected highly specific primers make it possible to identify the causative agent of cholera, to determine the biovar, serogroup and toxigenicity.

Thus, the proposed method and kit have a novelty: specific primers are selected to limit portions of the Vibrio cholerae genome: the wbeN fragment of the gene responsible for the synthesis of 01 antigen, the hlyA gene fragment, which is characteristic only of cholera vibrios of the biovar eltor, ctxA gene encoding the cholera toxin A subunit ; the optimal composition of the reaction mixture to perform multilocus PCR; optimal annealing conditions, which allows to simultaneously identify the causative agent of cholera, determine the biovar, serogroup and toxigenicity, as well as significantly reduce the analysis time, achieve a high level of sensitivity and specificity.

Method and kit for detection and characterization of cholera pathogen by multilocus polymerase chain reaction Table 1 Amplification program Matrix-controlled amplifiers: type "MJ Reseachr" Amplifiers with active regulation (in vitro): type "Tertsik" Temperature ° C Time Cycles Temperature ° C Time Cycles T 95 Pause 95 Pause 2 95 5 minutes one 95 5 minutes one T 95 40 s 5 95 10 s 5 t 62 40 s 62 10 s 5 72 40 s 72 10 s 6 95 30 s thirty 95 7 s thirty 60 30 s 60 7 s 72 30 s 72 7 s 72 5 minutes one 72 5 minutes one four 10 Storage 10 Storage

Claims (3)

1. A method for detecting and determining the biotype, serogroup, and toxigenicity of a cholera pathogen by the multilocus polymerase chain reaction (PCR) method, including DNA extraction from biological material and environmental objects, PCR staging in one step using three pairs of synthesized primers:
to the wbeN site of the wbf gene cluster encoding the synthesis of 01 - antigen,
wbeN1 - 5'-ATCAGTTTGCTTGCGCTTCTCC-3 ',
wbeN2 - 5'-ATCTGGACATCGCATCGCAC-3 ',
providing the formation of a fragment of 420 bp in size,
to the ctxA region of the gene encoding the synthesis of subunit A of cholera enterotoxin,
ctx2 - 5'-GGAGCCGGCATTCATCTGAA-3 ',
ctx3 - 5'-TTTCGGGCCATCTCCACTGA-3 ',
providing the formation of a fragment of 564 bp in size,
to the site of a specific for cholera vibrios biovar eltor hlyA gene encoding the synthesis of hemolysin,
hly1 - 5'-CGGGCAGATTCTAGACCTG-3 ',
hly2 - 5'-CGATGATCTTGGAGCATTCCCAC-3 ',
providing the formation of a fragment of 264 bp in size,
annealing the primers at a temperature of 95-60 ° C for 13.5 min with the number of amplification cycles equal to 35, gel electrophoresis, analysis of the PCR results on the obtained electrophoregram, followed by assessment of the detection results and determination of the presence or absence of specific amplified DNA bands on the electrophoregram in comparison with positive and negative control samples. 2. The kit for implementing the method according to claim 1, comprising components for DNA isolation, components for PCR, components for analyzing the results of PCR, the components are separated by 20% paraffin into two reaction mixtures, one of which contains 5 μl of oligonucleotide primers wbeN1 5'-ATCAGTTTGCTTGCGCTTCTCC-3 'and wbeN2 5'ATCTGGACATCGCATCGCAC-3', hly1 5'-CGGGCAGATTCTAGACCTG-3 'and hly2 5'-CGATGATCTTGGAGCATTCCCAC-3', ctx2 5'-GGAGCCGGCATTCATCTGAA-3 'and ctx3 5'-TTTCGGGCCATCTCCACTGA -3 'in a ratio of 1.5: 1.2: 1 and a dNTP solution, and the other 10 μl of a 2-fold buffer into which the required amount of Taq polymerase enzyme is introduced ex tempore. 3. The kit according to claim 2, characterized in that the buffer further comprises bromophenol blue and ficoll 400.

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