RU2560280C2 - Method of simultaneous identification of toxigenic strains of genovariants of el tor cholera causative agent and their differentiation on epidemic potential by method of multiplex polymerase chain reaction - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method is characterised in that the PCR is carried out in a single step in two reaction mixtures, each comprising a specially selected combination of primers: first - to fragments of genes rfbO1, rfbR, rtxC, ctxA, second - to fragments of genes ctxB^Class, vc0502, vc0514. The strains with low epidemic potential are characterised with the presence of amplicons of the genes rfbO1, rstC, ctxA, ctxB^Class, vc0502, vc0514. The strains with high epidemic potential are characterised with the presence of amplicons of the gene fragments rfbO1, rstC, ctxA, ctxB^Class and vc0514.

EFFECT: invention enables to identify quickly and reliably the toxigenic strains of V cholerae O1 genovariants of the biovar El Tor and to differentiate them based on the analysis of the structure of the pandemicity island VSP-II to the strains with low and high epidemic potential.

2 cl, 1 dwg, 3 tbl, 1 ex

Description

The invention relates to the field of medical microbiology, in particular to the identification of toxigenic strains of Vibrio cholerae O1 genovariants of the El Tor biovar serogroup and their differentiation by epidemic potential, and can be used in medical research institutions and Rospotrebnadzor services.

The epidemiological situation in the world of cholera - an acute, especially dangerous infectious disease, remains tense, the 7th pandemic continues. As you know, pandemic and epidemic cholera can cause only toxigenic strains of V. cholerae O1 (classical and El Tor biovars) and O139 serogroups. So, the causative agents of the current, 7th pandemic of cholera, which began in 1961 in Southeast Asia, are typical toxigenic strains of V. cholerae O1 serogroup El Tor biovar. Having a high epidemic potential at the time of occurrence, which is characterized as the ability to quickly penetrate the territory of many countries and cause epidemics there, El Tor vibrioes completely replaced the causative agent of the previous 6th pandemic - V. cholerae of the classic biovar from endemic territory and spread to many countries of Asia, Africa and Latin America. However, the ongoing evolutionary transformations of the El Tor vibrios and their acquisition of the classical biovar cholera vibrio genes (ctxB gene from the ctxAB operon encoding the cholera toxin biosynthesis) led to the appearance in 1992 of genetically modified strains or genovariants of V. cholerae El Top biovar [9]. Genovariants were characterized by increased virulence and had a higher epidemic potential than the typical El Tor vibrios, whose epidemiological potential decreased. As a result, genovariants have already replaced the typical El Tor strains of vibrios and quickly spread throughout the world. It should be noted that against the backdrop of the 7th pandemic in the same 1992, outbreaks of cholera caused by cholera vibrio O139 serogroups occurred in India. The rapid spread of these vibrios has raised concerns about the onset of the 8th pandemic. However, at present, O139 serogroup cholera vibrios circulate only in India, and El Tor vibrios are dominant.

The importation into new territories, pollution of water bodies, coexistence in the external environment with other microorganisms, etc., contributed to further transformations of the genome of the V. cholerae biovar El Tor genome. In the modern period, the cause of cholera is new strains of genovariants, one of the genetic features of which is the presence of an extensive deletion (21 genes) in the central part of the island of pandemic VSP-II (VSP-IIΔvc0495-vc0512). The global spread of VSP-II genovariant strains with altered structure and their displacement of previously emerged genovariants with intact VSP-II indicate a decrease in the epidemiological potential of the latter and a higher epidemic potential in new genovariant strains [2, 12].

Thus, strains of V. cholerae O1 genovariants of the El Tor biovar serogroup with different genome structures and different epidemic potential are currently circulating in the world, which can cause damage to the health of the population and the economy of the country to different degrees. Considering that strains of genovariants have already been imported into the Russian Federation since 1993, causing both single cases of the disease and outbreaks of cholera, in order to increase the effectiveness of the epidemiological surveillance system, it is necessary to develop a quick and reliable method for identifying toxigenic strains of genovariants and determining their epidemic potential.

A comprehensive gene and immunodiagnostic test system is known, which makes it possible to identify natural strains of cholera vibrio O1 and O139 of serogroups and determine their virulence [1]. However, this test system is not able to identify toxigenic gene variants of V. cholerae O1 El Tor biovar.

A known method for the differentiation of strains of V. cholerae O139 serogroups to toxigenic, epidemiologically significant and non-toxigenic, not having epidemic significance, isolates on alkyl sulfatase activity [5]. A known method for identifying epidemiologically significant strains of cholera vibrios V. cholerae eltor and V. cholerae O139 by their adhesive ability [3]. The described methods provide for the differentiation of epidemiologically significant strains of O139 serogroup and typical O1 strains of serogroup El Tor biovar based on the analysis of their phenotypic properties, the manifestation of which is a variable sign and may be lost during storage, and also depends on the quality of the media and reagents used. In addition, none of the above methods can detect toxigenic strains of V. cholerae genovariants El Tor biovar.

The invention is known according to patent WO2004020406, which describes a method for detecting strains of V. cholerae genovariants El Tor biovar based on the study of the structure of the tcpA and rstR genes [10]. However, the use of these target DNAs is not sufficient to identify strains of genovariants and does not provide for the possibility of their differentiation by epidemic potential.

A known method and test system for identifying toxigenic strains of Vibrio cholerae O1, determining their biovar and differentiating El Tor biovar strains into typical and changed by multiplex PCR in one step in two reaction mixtures [4]. The first reaction mixture contains primers for the rfbO1, cas3 and ctxB ^Class genes, and the second contains primers for the rfbO1, rtxC and ctxB Eltor ^genes . The invention allows to quickly and reliably determine the serogroup, biovar, toxigenicity of the studied strain and to differentiate the identified toxigenic strains of V. cholerae El Tor biovar into typical isolates and genovariants. However, this invention is not able to determine the epidemic potential of the identified genovariants, which is necessary to obtain the full characteristics of the identified V. cholerae isolate.

Thus, in this area there is an obvious need to develop a simple, reliable and fast method for the simultaneous detection of toxigenic strains of genetics of cholera vibrios of the El Tor biovar and their differentiation by epidemic potential.

The technical result consists in expanding the diagnostic capabilities of the method by simultaneously identifying the toxigenic genovariants of V. cholerae O1 serogroup El Tor biovar and their differentiation by epidemic potential.

The technical result is achieved due to the fact that the method of identification and differentiation of toxigenic strains of genovariants involves the isolation of the DNA of the studied strain, a one-step multiplex polymerase chain reaction in two reaction mixtures, each of which contains a specially selected combination of primers for the rfbO1, rfbR, rstC, ctxA genes in the first and ctxB ^Class , vc0502, vc0514 in the second, with an annealing temperature of primers 58 ° C for 10 sec with the number of amplification cycles equal to 25, followed by analysis of the amplified fragments of genes of the studied and control strains. The toxigenic strains of the V. cholerae el Tor biovar genovariants with a low epidemic potential are characterized by the presence of rfbO1, rstC, ctxA, ctxB ^Class amplicons, vc0502, vc0514 genes, the toxigenic genes of the V. cholerae El Tor biovar with a high epidemic potential determine amplicon fragments; rstC, ctxA, ctxB ^Class , vc0514.

Primers for regions of the rfbO1, rfbR, rstC, ctxA genes, and allele-specific primers for the ctxB ^Class gene were taken from literature [1, 6, 7, 8, 11]. Primers rfbO1-F - rfbO1-R to the gene regions from the rfbO1 cluster encoding the biosynthesis of the O1 antigen form 638 bp amplicon, rfbR-F - rfbR-R primers to the region of the rfbR gene that is part of the biosynthesis gene coding O139 antigen, form a PCR product with a size of 428 bp, primers rstC-F - rstC-R to the site of the rstC gene, which is part of the RS1 prophage, which is present only in the cholera vibrios of El Tor biovar, provide the formation of a 238 bp fragment ., primers ctxA-F - ctxA-R to the site of the ctxA gene encoding the A subunit of cholera toxin, provide specific amplification of a 564 bp DNA fragment; ctxB ^Class- F to ctxB ^Class- R primers on the ctxB gene allele of the classical type provide the formation of a 189 bp fragment.

The selection of DNA targets for determining the epidemic potential of genovariants was carried out on the basis of the analysis of the nucleotide sequences of the VSP-II pandemic island in toxigenic strains of the V. cholerae El Tor biovar genovariants isolated in the modern period of the 7th pandemic and containing different types of this genomic island. The vc0514 gene encoding a methyl-acceptor chemotaxis protein, the presence of which is characteristic of all types of VSP-II, was chosen as the first target. The vc0502 gene was selected as the second target, which encodes type IV drank, which is absent in strains with an extended deletion of this genomic island and having a high epidemic potential. The primers were designed using the Oligo 6.0 program based on the nucleotide sequences of the selected gene presented in the database and are highly specific. Primers VSPIIpilin-F - 5′-CTGTGATTCGGGCTTTATCGG-3 ′ and VSPIIpilin-R - 5′-GCGTAAACTGAGCCAATAAGC-3 ′ to the gene site vc0502 provide the formation of a fragment of 761 bp in size, primers VSPIIGAGGTAG-CTGGAG-FAG CTGGAG-FAG , VSPIIchem-R - 5′-CGATGAATAGCCTGTTGAAC-3 ′ to the vc0514 gene region provide the formation of a fragment of 604 bp

Primers for the vc0514, vc0502 genes are selected in such a way as to minimize the likelihood of their nonspecific annealing. The primers were tested for the possibility of the formation of hairpin structures with high melting points, as well as the formation of dimeric compounds of the same name as primers and unlike primers with each other. The size of the resulting fragments was selected to simplify the process of visualization and recording of results.

The optimization of the primer concentrations was achieved by mixing them in various ratios, as a result of the proposed concentration allow to obtain easily detectable amplicons. The concentration of each of the deoxynucleoside triphosphates dATP, dGTP, dTCP, dTTP is 2 mm. The qualitative and quantitative composition of the buffer for amplification was experimentally tested and are optimal for the study, the PCR formulation mode was determined.

The inventive identification method includes the following steps:

a) DNA isolation;

b) PCR;

c) analysis of the results.

DNA isolation

DNA is extracted from the cells using any set of reagents for DNA isolation (for example, “Set of reagents for DNA extraction” Reg. Ud. No. ФСР 2011 / 11172-280611 manufactured by RosNIPCHI “Microbe” containing an aqueous suspension of nucleosorbent, buffer 1, including guanidinezothiocyanate and EDTA, buffers 2, 3 - for washing and eluent for DNA (TE buffer).

PCR

PCR is carried out in one step according to the following program: preliminary denaturation at 96 ° C - 2 min, 25 cycles, including 96 ° C - 10 s, 58 ° C - 10 s, 72 ° C - 30 s, final chain completion at a temperature 72 ° C - 7 min 20 sec.

Results Analysis

The amplified DNA after PCR is detected by horizontal gel electrophoresis in a 2% agarose gel. The PCR results are taken into account by comparing the obtained amplicons with control samples in accordance with the identification table (Table 1).

The method is as follows.

1. Sample preparation is carried out according to MU 1.3.2569-09 “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV” in a class II biological safety box in type IV anti-plague suit in rubber or latex gloves. Vibrio cholerae cells grown on LB agar for 18 hours are resuspended in a 0.9% sodium chloride solution to a concentration of 10 ⁹ microbial cells in 1 ml and then diluted in deionized water to a concentration of 1 × 10 ⁷ m.k. / ml Then sodium merthiolate is added to the suspension to a final concentration of 1: 10000 (0.01%) and heated at a temperature of 56 ° C for 30 minutes. Next, 100 μl of the sample was transferred to a 1.5 ml microcentrifuge tube, a lysis solution prepared on the basis of 6M guanidine isothiocyanate was added, and incubated for 15 min at a temperature of 65 ° C. After performing these procedures, the material is considered disinfected.

2. DNA isolation of the studied strains of cholera vibrio is carried out using a set of reagents for DNA isolation. The DNA isolation kit is removed from the refrigerator and kept at room temperature. DNA isolation is carried out according to the instructions for the kit. The purified DNA is stored at + 4 ° C.

3. For PCR, the required number of microtubes is prepared corresponding to the number of samples tested, as well as six more for each reaction mixture: five differentiating positive controls and one negative control (deionized water).

PCR is carried out in a volume of 25 μl of the reaction mixture per 1 DNA sample. The compositions of the reaction mixture No. 1 and No. 2 are presented in table 2. To prevent evaporation of the reaction sample in the tubes carefully layered mineral oil on top. In the prepared mixture under oil contribute 10 μl of sample. 10 μl of deionized water are added to a test tube designated as a negative control, and 10 μl of DNA of the control strains, respectively, are added to test tubes with positive controls. The DNA of the toxigenic strain V. cholerae 569B O1 of the gray group of the classic biovar (rfbO1 ⁺ , rfbR ^- , rstC ^- , ctxA ⁺ , ctxB ^{Class +} , vc0502 ^- , vc0514 ^- ), a typical toxigenic strain of V. cholerae M738 O1 serogroup, is used as positive controls El Top biovar (rfbO1 ⁺ , rfbR ^- , rstC ⁺ , ctxA ⁺ , ctxB ^Class- , vc0502 ⁺ , vc0514 ⁺ ), a toxigenic strain of the genovariant V. cholerae M1266 El Top with an intact VSP-II pandemic island and low epidemic potential (rfbO ⁺ , rfbR ^- , rstC ⁺ , ctxA ⁺ , ctxB ^{Class +} , vc0502 ⁺ , vc0514 ⁺ ), a toxigenic strain of the gene variant V. cholerae M1509 El Top, containing VSP-II with an extended deletion and having a high e theidemic potential (rfbO1 ⁺ , rfbR ^- , rstC ⁺ , ctxA ⁺ , ctxB ^{Class +} , vc0502 ^- , vc0514 ⁺ ), toxigenic strain V. cholerae P16064 O139 serogroup (rfbO1 ^- , rfbR ⁺ , rstC ⁺ , ctxA ⁺ , ctxA ⁺ , vc0502 ⁺ , vc0514 ⁺ ).

Amplification of DNA is carried out using the programmable thermocycler “Tertzik” (DNA-Technology, Russia) at the following temperature conditions (according to the matrix method of regulation): denaturation temperature - 96 ° C for 2 min; 25 cycles — DNA denaturation at 96 ° C for 10 sec, primer annealing at 58 ° C — 10 sec, complementary chain synthesis at 72 ° C — 30 sec; final synthesis of the complementary chain at a temperature of 72 ° C for 7 min 20 sec. Amplification of control samples is carried out simultaneously with the test samples in the same device and under the same conditions.

4. To analyze the results of PCR, a 2% agarose gel is prepared. For this, 50 ml of TAE buffer is added to 1 g of agarose. The agarose is brought to a boil, cooled at room temperature to about + 65 ° C, 5 μl of ethidium bromide are added, the contents of the flask are gently mixed with uniform rotational movements and poured into a special bridge so that the gel thickness is about 4 mm. Then set the comb and leave to harden. After polymerization, the comb is carefully removed and the gel is transferred to the chamber for electrophoresis. To the resulting amplicons add 5 μl of sample buffer, mix with the same tip and add to the gel wells under the buffer solution. The camera is connected to a current source and a voltage of 7-10 V / cm is set. Electrophoretic separation is continued for 80 min until the leading dye passes about 4/5 of the track length (recommended track length is 10 cm), after which the gel is removed from the chamber and placed on a traisilluminator glass with UV radiation of 310 nm. Fragments of the analyzed DNA appear as orange-red bands.

The results are evaluated by comparing PCR amplicons with marker fragments of control V. cholerae strains. The results are taken into account in accordance with the identification table (Table 1).

Using the primers contained in the first reaction mixture, the serogroup of the studied strain V. cholerae - O1 (rfbO1 ⁺ ) or 0139 (rfbR ⁺ ), biovar (classic - rstC ^- , El Top - rstC ⁺ ) and toxigenicity (ctxA ⁺ ) are determined. The primers that make up the second reaction mixture allow the V. cholerae O1 strains of the El Tor biogar serogroup to be separated into typical isolates (ctxB ^Class- ) and genovariants (ctxB ^{Class +} ) and differentiate genovariants according to the structure of the VSP-II pandemic island into low strains (vc0502 ⁺ , vc0514 ⁺ ) and high (vc0502 ^- , vc0514 ⁺ ) epidemic potential.

In FIG. Figure 1 shows the electrophoregram of amplicons obtained by staging multiplex PCR with control strains of V. cholerae. Lane 1 shows the position of the amplicons of the toxigenic strain V. cholerae 569B O1 serogroup classic biovar; in lane 2 - a typical toxigenic strain of V. cholerae M738 O1 serogroup El Tor biovar, on lane 3 - of a toxigenic strain of genovariant V. cholerae M1266 O1 serogroup El Tor with intact pandemic island VSP-II and low epidemic potential, on lane 4 - toxigenic genovariant strain V. cholerae M1509 O1 serogroup El Topovar, carrying VSP-II with an extended deletion, and having a high epidemic potential, on track 5 - toxigenic strain V. cholerae P16064 O139 serogroup. Lane 6 - negative control.

The specificity of the proposed method is confirmed through the use of strains of closely related species - V. mimicus, V. anguillarum, V. parahaemolyticus, as well as enterobacteria - E. coli, S. enteritidis, Sh. flexneri. The results of PCR testing of these strains were negative, which indicates the specificity of the method.

The invention is illustrated by the following example.

Example 1. To determine the effectiveness of the proposed method investigated 28 clinical strains of V. cholerae isolated in different territories and in different years.

In 22 studied strains, amplicons of 638 bp were detected. and 564 bp, indicating their belonging to the toxigenic O1 strains of the serogroup, two strains (V. cholerae 14300 and M14380) were atoxigenic, since they lacked an amplicon of 564 bp in size. (table 3). In addition to the rfbO1 gene, in 18 strains of V. cholerae, in addition to the rfbO1 gene, a biovar-specific rstC gene was detected using reaction mixture No. 1, since an amplicon of 238 bp was identified, which indicates their belonging to the El Tor biovar O1 toxigenic vibrio. Moreover, the ctxB ^Class gene is absent in 6 strains (an amplicon of 189 bp in reaction mixture No. 2), as is the case of strain M738, taken as a control, which indicates their relation to typical toxigenic strains of V. cholerae O1 El biovar Thor (Fig 1., lane 2, table 3). At the same time, the presence of this gene was found in the remaining 12 isolates, which proves their belonging to the toxigenic genovariants of V. cholerae 01 El Tor biovar. When analyzing the structure of the VSP-II pandemic island, this group of strains was divided into two. The first consisted of 7 strains that contained two fragments of 761 bp in the reaction mixture No. 2 and 604 bp (table 3). These genovariant strains have low epidemic potential. At the same time, in the second group, including 5 isolates, only a fragment of 604 bp was amplified, as in the control V. cholerae M1509 containing VSP-II with an extended deletion (Fig. 1, lane 4, Table 3). These strains, isolated in recent years (2004-2012), are genovariants with high epidemic potential, which have practically replaced genovariants with low epidemic potential in many regions of the world.

In addition, using this PCR, it is also possible to identify O1 strains of the serogroup of the classic biovar and O139 strains of the serogroup. So, among the isolates studied by us, five strains were identified as toxigenic strains of the classic biovar. The amplification of DNA fragments of 638 and 564 bp in size occurred in these strains. in the reaction mixture No. 1 and 189 bp in the reaction mixture No. 2, which indicates the presence of rfbO1, ctxA and ctxB ^Class genes in their genome (Fig. 1., lane 1, table 3). According to the presence of amplicons in sizes 428, 238 and 564 bp in the reaction mixture No. 1 and 761, 604 bp in the reaction mixture No. 2, which indicates the presence of the rfbR, rstC, ctxA, vc0502, vc0514 genes, respectively, the five studied strains were assigned to the toxigenic strains of V. cholerae O139 serogroup (Fig. 1., lane 5, table 3).

Thus, the developed method makes it possible to quickly and reliably identify toxigenic strains of V. cholerae O1 genovariants of the El Tor biovar serogroup that carry the classical type ctxB gene in the genome and differentiate them based on the analysis of the structure of the VSP-II pandemic island into genes with high and low epidemic potential .

Bibliography

1. A comprehensive gene and immunodiagnostic test system for the identification of cholera vibrios O1 and O139 serogroups and assessment of their virulence. Patent for invention No. 2404257, published November 20, 2010

2. Smirnova NI, Zadnova SP, Agafonov DA., Shashkova AV, Cheldyshova NB, Cherkasov AV Comparative molecular genetic analysis of the mobile elements of natural strains of the cholera pathogen. 2013; 49 (9): 1036-1047.

3. A method for identifying the epidemically significant cholera vibrios of Vibrio cholerae eltor and Vibrio cholerae 0139 by their adhesive ability. RF patent No. 2332460, published August 27, 2008

4. A method for identifying toxigenic strains of V. cholerae O1, determining their biovar and differentiating the biovar eltor strains into typical and modified multiplex polymerase chain reaction and test system for its implementation. RF patent No. 2458141, published on 08/10/2012.

5. A method for differentiating strains of Vibrio cholerae O139 serogroup by alkyl sulfatase activity. RF patent No. 2473697, published January 21, 2013.

6. Fields P.I., Popovic T., Wachsmuth K., Olsvik O. Use of polymerase chain reaction for detection of toxigenic Vibrio cholerae O1 strains from the Latin American cholera epidemic. J. Clin. Microbiol. 1992; 30: 2118-2121.

7. Goel A.K., Ponmariappan S., Kamboj D.V., Singh L. Single multiplex polymerase chain reaction for environmental surveillance of toxigenic-pathogenic O1 and non-O1 Vibrio cholerae. Folia Microbiol. 2007, 52: 81-85.

8. Morita M., Ohnishi M., Arakawa E. et al. Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype E1 Tor. Microbiol. bnmunol. 2008, 52 (6): 314-317.

9. Nair G. B., Faruque S. M., Bhuiyan N. A. et al. New Variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh. J. Clin. Microbiol. 2002, 40 (9): 3296-3299.

10. Variants of Vibrio cholerae O1 biotype El Tor with attributes of classical biotype. Invention WO 2004020406, publication 11/03/2004.

11. Waldor MK, Rubin EJ, Pearson GDN et al. Regulation, replication, and integration functions of the Vibrio cholerae CTX _φ are encoded by region RS2. Mol. Microbiol. 1997, 24 (5): 917-926.

12. Taviani E., Grim C. J., Choi J. et al. Discovery of novel Vibrio cholerae VSP-II genomic islands using comparative genomic analysis. FEMS Microbiol. Lett 2010; 308: 130-137.

Claims (2)

1. A method for the simultaneous identification of toxigenic strains of genes of the El Tor cholera pathogen and their differentiation by epidemic potential by the method of multiplex polymerase chain reaction, characterized in that the polymerase chain reaction is carried out in one step in two reaction mixtures, each of which contains a specially selected combination of primers for the genes rfbO1, rfbR, rstC, ctxA in the first, and ctxB ^Class , vc0502, vc0514 in the second, with annealing temperature of primers 58 ° C for 10 sec with the number of amplification cycles equal to 25, followed by analysis of the amplified fragments of the genes of the studied and control strains; for toxigenic strains of V. cholerae O1 genovariants of the El Tor biovar with low epidemic potential, amplicons of rfbO1, rstC, ctxA, ctxB ^Class , vc0502, vc0514 genes are characteristic; for toxigenic genariates of V. cholerae O1 El Tor biovar with high epidemic potential are determined rfbO1, rstC, ctxA, ctxB ^Class , vc0514. 2. The method according to p. 1, characterized in that the primers designed for sections of the vc0502, vc0514 genes have the following nucleotide sequences:
VSPIIpilin-F - 5′-CTGTGATTCGGGCTTTATCGG-3 ′, VSPIIpilin-R - 5′-GCGTAAACTGAGCCAATAAGC-3 ′, VSPIIchem-F - 5′-CTTGATGGAGCGGAGAAAAC-3 ′ VSPIIAGT-5GTGAGTGA-3GTGAGTGA-3GTGATGTGA-3GTGAGTGA-3GTGATGTGA-3G.