RU2815711C2 - Method for identification of toxigenic genetic variants of cholera causative agent el tor with set of mutations in genes of virulence and pandemic island genes by multiplex polymerase chain reaction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medical microbiology, in particular to identification of new versions of toxigenic strains of Vibrio cholerae biovar El Tor, isolated from various sources. Method for identifying toxigenic genetic variants of cholera causative agent El Tor with a set of mutations in virulence genes and VSP-II island genes by multiplex polymerase chain reaction involves preparation and disinfection of analyzed samples, DNA extraction and multiplex PCR in one step in two reaction mixtures with subsequent identification of toxigenic novel variants by the presence of amplicons to five targets - ctxA, ctxB7, tcpACIRS101, rtxA4, vc0514 and absence of amplicon to target vc0502. First of the reaction mixtures contains a specially selected combination of primers SEQ ID NO: 1, 2, 6 on target DNA ctxA, ctxB7, tcpACIRS101. Second mixture contains SEQ ID NO: 3, 4, 5 on DNA target rtxA4, vc0502 and vc0514.

EFFECT: invention implementation provides higher efficiency of detection of new toxigenic genetic variants of cholera causative agent El Tor with high virulence and high epidemic potential.

1 cl, 1 dwg, 5 tbl, 2 ex

Description

The invention relates to the field of medical microbiology, in particular to the identification of new variants of toxigenic strains of Vibrio cholerae biovar El Tor, isolated from various sources.

The epidemiological situation with cholera in many endemic regions of Asia, Africa and America is unfavorable, which creates real risks of interstate and cross-border import of this particularly dangerous infection into the territory of the Russian Federation.

Toxigenic strains of Vibrio cholerae serogroup O1 biovar El Tor, which caused three waves of the current, 7th, cholera pandemic, which began in 1961 and continues to this day, include typical, genetically modified (or atypical strains) and new genetic variants of the pathogen , differing in virulence and epidemic potential. The main difference between typical and atypical strains of the cholera pathogen isolated during the first (1962-1991) and second waves (1991-2004) of the pandemic, respectively, are different alleles of key pathogenicity genes. Typical strains have alleles ctxB3 and tcpA ^{El Tor} , atypical - ctxB1 - tcpA ^{El Tor} or ctxB1 - tcpA ^CIRS101 , encoding the B subunit of cholera toxin and the main subunit of toxin-coregulated pili, a key factor in vibrio colonization of the small intestine. In addition, a number of strains carry a pandemic island VSP-II, modified due to the deletion of several genes, associated with the ability of the pathogen to spread epidemically. New genetic variants that arose during the evolution of the third wave (2006 to the present) are characterized by previously unknown mutations in the pathogenicity genes ctxB (ctxB7 allele) and rtxA (rtxA4 allele), encoding the B-subunit of cholera toxin and the cytotoxin MARTX, in combination with an extended deletion of the pandemic island genes VSP-IIΔ(0495-0512) and the tcpA ^CIRS101 allele. Due to new mutations, the main markers of such strains were the alleles of the virulence genes ctxB7, tcpA ^CIRS and rtxA4, as well as the pandemic island VSP-IIΔ(0495-0512) with an extended deletion. These changes have led to a significant increase in their pathogenic and epidemic potential.

Currently, despite the dominance of new variants, toxigenic cholera vibrios circulating in endemic foci of cholera and introduced into the territory of the Russian Federation are represented by a diverse group, consisting of both previously emerged atypical strains and strains with new mutations of epidemically significant genes. The real risk of importation of Vibrio cholerae into Russia with a different set of altered virulence and epidemic genes, capable of causing various damage to the health of the population and the country's economy, indicates the need to develop a method for identifying new genetic variants of V. cholerae O1 biovar El Tor in order to obtain reliable information about the genetic properties of the detected strains in order to predict the epidemiological situation of cholera.

A complex geno- and immunodiagnostic test system is known for identifying Vibrio cholerae O1 and O139 serogroups and assessing their virulence, which allows the identification of natural strains of Vibrio cholerae O1 and O139 serogroups and determining their virulence [1].

A multiplex PCR test system is known using primers for the species-specific hapA gene and the main virulence genes ctxA, tcpA and toxR, which allows simultaneous identification of V. cholerae strains and differentiation of them according to epidemic significance [2].

The described methods and test systems are intended for the detection and identification of Vibrio cholerae strains, determination of their serogroup and biovar, differentiation of typical strains of V. cholerae biovar El Tor according to epidemic significance, however, they are not capable of identifying new variants of atypical toxigenic strains of the causative agent of the current cholera pandemic.

There is a known method and test system for identifying toxigenic strains of Vibrio cholerae O1, determining their biovar and differentiating V. cholerae biovar El Tor into typical and modified variants using the PCR method [3]. According to this method, multiplex PCR is carried out in one step in two reaction mixtures, each of which contains a specially selected combination of primers: one for the rfbO1, cas3 and ctxB ^Class genes, the second for the rfbO1, rtxC and ctxB ^Et genes. The invention makes it possible to quickly and reliably identify the biovar of cholerae vibrios O1 serogroup, determine their toxigenicity and differentiate the identified toxigenic strains of V. cholerae biovar El Tor into typical and atypical.

There is a known method for differentiating strains of V. cholerae O139 serogroup by alkylsulfatase activity, which makes it possible to confirm the toxigenicity of the strain and its epidemiological significance [4].

A method for identifying epidemically significant cholera vibrios V. cholerae El Tor and Vibrio cholerae O139 by their adhesive ability to detect epidemically significant, toxigenic and atoxigenic hemolytic strains is described [5].

There is a known method that makes it possible to identify toxigenic strains of genovariants of V. cholerae O1 biovar El Tor and differentiate them based on analysis of the structure of the VSP-II pandemic island into strains with low and high epidemic potential using the multiplex polymerase chain reaction method [6].

There is a known method for identifying Vibrio cholerae O1 strains, which makes it possible to determine their toxigenicity and biovar with differentiation of the ElTor biovar into typical and genetically modified variants using the multiplex polymerase chain reaction method, taking into account the results in “real time” [7].

However, these methods do not ensure the identification of new variants of atypical strains containing a set of altered genes associated with pathogenic and epidemic potential, which is necessary for improving methods of molecular epidemiological monitoring of the cholera pathogen and predicting the epidemiological situation.

Thus, there is a clear need in the field to develop an informative and rapid method for identifying toxigenic novel genetic variants of V. cholerae biovar El Tor.

The objective of the invention is to select DNA targets in the genome of the causative agent of cholera that determine whether the studied strain of V. cholerae belongs to new toxigenic genetic variants, to select the optimal combination of oligonucleotide primers and polymerase chain reaction parameters, which makes it possible to effectively identify new variants of the pathogen.

The technical result of the claimed invention is to increase the efficiency of identifying new toxigenic genetic variants of the cholera pathogen El Tor with increased virulence and high epidemic potential.

The technical result is achieved by a method for identifying toxigenic new genetic variants of the causative agent of cholera El Tor using the multiplex polymerase chain reaction method, which involves isolating the chromosomal DNA of the strain under study, conducting a one-stage multiplex polymerase chain reaction in two reaction mixtures, the first of which contains a specially selected combination of primers for the ctxA genes, ctxB7, tcpA ^CIRS101 , and the second - rtxA, vc0502, vc0514, with a sequential decrease in the primer annealing temperature from 58 to 54°C with an interval of 2°C every five amplification cycles with a total number of amplification cycles equal to 35 (Table 2), s subsequent electrophoretic analysis of the obtained amplified gene fragments of the studied and control strains.

The sequences of primers ctxA-F - ctxA-R, tcpA-F2 - tcpA-Rev, rtxA4-F - rtxA4-R2, vc0502-F - vc0502-R, vc0514-F - vc0514-R, ctxB7-F3 were taken from literature sources, the ctxB7-R primer was designed by the authors to optimize the amplicon size for better separation of amplification products in the gel.

Primer pairs used in the claimed method:

ctxA-F - 5'-CGGGCAGATTCTAGACCTCCTG-3'

ctxA-R - 5'-CGATGATCTTGGAGCATTCCCAC-3' - primers for the ctxA gene, encoding the synthesis of subunit A of cholera toxin, providing amplification of a fragment of 564 bp. [8];

ctxB7-F3 - 5'-GTTTTACTATCTTCAGCATATGCGA-3'

ctxB7-R - 5'-ATCGCATGAGGCGTTTTTATT-3' - primers for the ctxB7 allele (C⇒A p: 59 from the beginning of the gene) of the ctxB gene, encoding the synthesis of subunit B of cholera toxin, with the help of which a PCR product of 300 bp in size is formed. [9];

tcpA ^CIRS101 -F2 - 5'-CCAGCTACGCGCAAACGCAGG-3'

tcpA ^CIRS101 -Rev - 5'-CCGACTGTAATTGCGAATGC-3' - primers for the tcpA ^CIRS allele (A⇒G p: 266 from the beginning of the gene) of the tcpA gene, encoding the main subunit of toxin-corrected adhesion pili, provide the formation of a fragment whose length is 167 bp .n. [10];

rtxA4-F - 5'-TACTTTAATGGTAACCGCGCT-3'

rtxA4-R2 - 5'-TGTGAACCACGTCTGCT-3' - primers for the rtxA4 allele (G⇒A p: 13602 from the beginning of the gene) of the rtxA gene, responsible for the synthesis of RTX toxin in V. cholerae biovar El Tor, provide specific amplification of a DNA fragment of 187 bp [eleven];

vc0502 - 5'-CTGTGATTCGGGCTTTATCGG-3'

vc0502-R - 5'-CTTGATGGAGCGGAGAAAAC-3' - primers for the vc0502 gene encoding type IV pili, with the help of which a fragment of 761 bp is formed. [12];

vc0514-F - 5'-CTTGATGGAGCGGAGAAAAC-3'

vc0514-R - 5'-CGATGAATAGCCTGTTGAAC-3' - primers for the vc0514 gene, encoding the synthesis of the methyl acceptor chemotaxis protein, ensuring the formation of a fragment of 604 bp. [12];

The optimal concentrations of primers in the reaction mixtures were determined experimentally. Deoxynucleotides dATP, dGTP, dCTP and dTTP are taken in equimolar ratio to achieve a final concentration of 2 mM each in the dNTP stock solution. The optimal ratio of other components for each of the two reaction mixtures and a common amplification program for them were experimentally selected.

The method is carried out as follows.

1. DNA isolation is carried out using any set of reagents for DNA isolation by nucleosorption in the presence of guanidine isothiocyanate (for example, “Set of reagents for DNA isolation”, registration number FSR 2011/11172-280611 produced by RosNIPCI “Microbe”, containing an aqueous suspension of nucleosorbent , buffer 1, including guanidine isothiocyanate and EDTA, buffers 2, 3 - for washing and eluent for DNA (TE buffer). The DNA extraction kit is removed from the refrigerator and kept at room temperature. DNA extraction is carried out according to the instructions for the kit. Purified DNA is stored at a temperature of +4°C.

2. To carry out PCR, prepare the required number of microtubes corresponding to the number of samples being tested, as well as six more for each reaction mixture: four differentiating positive controls and one negative control (deionized water). The polymerase chain reaction is carried out in a reaction mixture volume of 25 μl per 1 DNA sample. The composition of reaction mixtures No. 1 and No. 2 are presented in Table 1. 10 μl of sample is added to the prepared mixture. 10 μl of deionized water is added to a test tube designated as a negative control, and 10 μl of DNA from control strains is added to test tubes with positive controls, respectively. DNA from a toxigenic new variant of V. cholerae (ctxA ⁺ ctxB7 ⁺ tcpA ^CIRS101+ rtxA4 ⁺ vc0502 ^- vc0514 ⁺ ) and a toxigenic typical/atypical strain (ctxA ⁺ ctxB7 ^- tcpA ^CIRS101- rtxA4 ^- vc0502 ⁺ vc0514 ⁺ ) are used as positive controls.

DNA amplification is carried out using a programmable thermostat, for example “Tertsik” (DNA-Technology, Russia) or similar. PCR is carried out in one stage according to the following program: preliminary denaturation at a temperature of 96°C - 2 min, 5 cycles, including 96°C - 10 s, 58°C - 10 s, 72°C - 30 s; 5 cycles including 96°C - 10 s, 56°C - 10 s, 72°C - 30 s; 25 cycles, including 96°C - 10 s, 54°C - 10 s, 72°C - 30 s; final completion of the chain at a temperature of 72°C - 2 minutes (Table 2).

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

The results of PCR analysis are recorded by comparing the obtained amplicons with control samples in accordance with the identification table (Table 3).

Using the primers contained in the first reaction mixture, the toxigenicity (presence of the ctxA ⁺ gene) of the studied strains of V cholerae O1 biovar El Tor is determined and differentiation is carried out into typical/atypical (ctxB7 ^- tcpA ^CIRS101- /ctxB7 ^- tcpA ^CIRS101+ ) and new variants (ctxB7 ⁺ tcpA ^CIRS101+ ). The primers included in the second reaction mixture reveal the presence in the studied strains of marker genes characteristic of new variants: the rtxA4 allele, the vc0514 gene, as well as the absence of the vc0502 gene.

The belonging of the V. cholerae O1 biovar El Tor strains to the new variant is determined by the total presence/absence of amplicons with primers for the 6 DNA targets under study.

Based on the presence of amplicons with primers for the targets ctxA, ctxB7, tcpA ^CIRS101 , rtxA4, vc0514, but the absence of those for the vc0502 gene, the strain belongs to a new variant of the pathogen. Other toxigenic strains are characterized by the presence of amplicons with primers to the ctxA gene, but not to ctxB7, and different combinations of amplicons to other target genes (Table 3).

In fig. Figure 1 shows an electropherogram of amplicons obtained by performing multiplex PCR with control strains of V. cholerae. Lanes 1 and 6 show the position of the amplicons of the toxigenic strain L-3226 of the new variant of V. cholerae O1 biovar El Tor, with the virulence genes ctxA, ctxB7, tcpA ^CIRS101 , rtxA4 and the vc0514 gene of the deleted VSP-II. In lanes 2 and 7 - toxigenic strain M1293 of V. cholerae O1 biovar El Tor, containing the ctxA, and vc0502 and vc0514 genes from intact VSP-II, but lacking the ctxB7, tcpA ^CIRS101 and rtxA4 alleles. In lanes 3 and 8 - toxigenic strain M1429 of V. cholerae 01 biovar El Tor, containing the ctxA, tcpA, rtxA4 and vc0514 genes, but not having the ctxB7 allele of the ctxB gene, as well as vc0502. In lanes 4 and 9 - a toxigenic strain of the new variant 3265/80 of V. cholerae 01 biovar El Tor, with the virulence genes ctxA, ctxB7, tcpA ^CIRS101 , rtxA4 and the vc0514 gene of the deleted VSP-II. Lane 5 - negative control (deionized water). The results obtained were confirmed by performing PCR with single primers, followed by fragmentary Sanger sequencing of the resulting amplification products [13].

The effectiveness of the proposed method for identifying new variants was confirmed on the basis of PCR analysis of 35 natural clinical isolates (Table 4), as well as the use of additional research methods (comparative analysis of the results of whole-genome sequencing of these strains).

When testing DNA isolated from cultures of bacteria other than the Vibrio cholerae species, specific amplification products are not formed, which is reflected in the absence of visible amplicons in the electropherogram. The specificity of the proposed method was confirmed by the results of PCR analysis of 22 strains of V. cholerae O139 serogroup, closely related species - Vibrio mimicus, Vibrio anguillarum, Vibrio parahaemolyticus, as well as enterobacteria - Escherichia coli, Salmonella enteritidis, Shigellaflexneri. The results of PCR testing of these heterologous strains are presented in table. 5

The invention is illustrated by the following examples:

Example 1. Study of a culture suspected of belonging to V. cholerae grown on a solid nutrient medium.

For the study, use a culture suspected of belonging to the species V. cholerae, grown on a solid medium suitable for the cultivation of Vibrio cholerae (for example, 1.5% LB agar, pH 7.6) at 37°C for 18 hours. Next, a suspension of microbial cells is prepared in physiological solution to a concentration of 10 ⁹ microbial cells in 1 ml, followed by dilution in deionized water to a concentration of 1 × 10 ⁷ microbial cells/ml. Afterwards, the test sample is disinfected in accordance with MU 1.3. 2569-09. DNA extraction of the V. cholerae strain under study is carried out using commercial DNA extraction kits. The resulting DNA is used for PCR using specific primers for target DNA ctxA, ctxB7, tcpA ^CIRS101 , rtxA4, vc0502 and vc0514. Formation of amplicons with sizes of 167 bp, 300 bp, 564 bp on the electropherogram. in reaction mixture No. 1 and 187 bp, 604 bp. in reaction mixture No. 2 indicates the presence of the ctxA, ctxB7, tcpA ^CIRS101 , rtxA4 and vc0514 genes in the genome of the strain under study, but the absence of the vc0502 gene. It is this set of tested genes that is typical for new genetic variants (Fig. 1, Table 3, Table 4). Thus, these results indicate that the studied strain belongs to the new toxigenic variants of V. cholerae 01 serogroup of biovar El Tor.

Example 2. Study of a broth culture of a collection strain of the causative agent of cholera.

The test strain is inoculated into a test tube with a liquid medium suitable for growing Vibrio cholerae (for example, LB broth) and incubated for 18 hours at 37°C. 100 μl of the resulting culture is selected and the DNA preparation is prepared and multiplex PCR is carried out similarly to example No. 1. Interpretation of PCR results is carried out according to Table 3. The presence in the genome of the studied strain of a set of alleles (ctxA ⁺ ctxB7 ⁺ tcpA ^CIRS101+ rtxA4 ⁺ vc0502 ^- vc0514 ⁺ ) indicates that it belongs to the toxigenic new genetic variants of V. cholerae O1 serogroup of biovar El Tor (Fig. 1 , table 3, table 4).

Bibliography

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List of primer sequences

SEQ ID NO: 1

- ctxB7-F3 - 5'-GTTTTACTATCTTCAGCATATGCGA-3',

- ctxB7-R - 5'-ATCGCATGAGGCGTTTTTATT-3',

SEQ ID NO: 2

- tcpA-F2 - 5'-CCAGCTACGCGCAAACGCAGG-3'

- tcpA-Rev - 5'-CCGACTGTAATTGCGAATGC-3',

SEQ ID NO: 3

- rtxA4-F - 5'-TACTTTAATGGTAACCGCGCT-3',

- rtxA4-R2 - 5'-TGTGAACCACGTCTGCT-3',

SEQ ID NO: 4

- vc0502-F - 5'-CTGTGATTCGGGCTTTATCGG-3',

- vc0502-R - 5'-CTTGATGGAGCGGAGAAAAC-3',

SEQ ID NO: 5

- vc0514-F - 5'-CTTGATGGAGCGGAGAAAAC-3',

- vc0514-R - 5'-CGATGAATAGCCTGTTGAAC-3',

SEQ ID NO: 6

- ctxA-F - 5'-CGGGCAGATTCTAGACCTCCTG-3',

- ctxA-R - 5'-CGATGATCTTGGAGCATTCCCAC-3'

Claims (1)

A method for identifying toxigenic genetic variants of the cholera causative agent El Tor with a set of mutations in virulence genes and pandemic island genes VSP-II using the multiplex polymerase chain reaction method, involving the preparation and disinfection of test samples, DNA isolation and multiplex PCR in one step in two reaction mixtures, the first of which contains a specially selected combination of primers SEQ ID NO: 1, 2, 6 on target DNA ctxA, ctxB7, tcpA ^CIRS101 , the second mixture includes SEQ ID NO: 3, 4, 5 on target DNA rtxA4, vc0502 and vc0514, with subsequent identification of toxigenic new variants by the presence of amplicons to five targets - ctxA, ctxB7, tcpA ^CIRS101 , rtxA4, vc0514 and the absence of an amplicon to the target vc0502.