RU2473701C1 - Set and method for accelerated plague microbe identification and simultaneous differentiation of virulent and avirulent strains of ypestis by plasmid profiling thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: set involves primers and fluorescent-marked probes for 3a, cafl, pla, irp2, hmsH and IcrV gene sites within two different PCR mixtures. The set is used for the purpose of accelerated plague microbe identification and simultaneous differentiation of virulent and avirulent strains of Y.pestis by plasmid profiling. A method with the use of the set provides conducting multiplex PCR in two test tubes and test recording by probe mark signals.

EFFECT: invention enables higher diagnostic effectiveness of the strains.

2 cl, 8 dwg, 10 tbl, 1 ex

Description

The invention relates to the field of biotechnology and can be used in the diagnosis of the plague pathogen, namely, to identify strains of the plague pathogen, as well as for solving research problems.

Currently, the epidemiological situation of plague throughout the world remains quite complex. This is due to the activity of natural foci of plague, the possibility of introducing pathogens from endemic territories to epidemically safe ones due to human economic activity. In addition, Y. pestis is classified as the most dangerous of the possible agents of bioterrorism due to its high contagiousness and mortality [1]. Therefore, a quick and clear diagnosis of the plague pathogen will make it possible to quickly assess the epidemic situation and to carry out antiepidemic measures in a timely manner in order to prevent the spread of infection.

Virulence of the plague pathogen is a polydeterminant trait that is controlled by a large number of genes localized on the chromosome and plasmids. On the plague microbe chromosome there is a pigmentation region (pgm region), which includes an hms locus encoding hemin accumulation proteins and a high pathogenicity island (AFP) with iron utilization genes. The genes of the pCad plasmid encode the synthesis of the LcrV protein (V antigen), Yop effector proteins that are part of the type III secretion system, whose action is aimed at suppressing the phagocytic activity of mammalian immune system cells. The most important factors for the pathogenicity of the plague pathogen are the products of gene expression of the chromosome island of high pathogenicity (AFP) and the ur operon. plasmids pCad. Loss of any of these markers leads to the loss of virulence by strains for laboratory animals.

To identify the selected cultures using microbiological, biochemical, biological methods. One of the main features studied in the identification of Y. pestis cultures is the determination of virulence, which is carried out using the biological method and plating on solid nutrient media with hemin. Such studies are time consuming and lengthy.

Currently, molecular genetic methods, especially the polymerase chain reaction (PCR), which has high sensitivity, specificity and allows you to get the result within 3-5 hours, are becoming more widespread. There are various ways to study the properties of the plague pathogen using PCR.

A known method for determining the loss of virulence by strains of the plague microbe due to the loss of the pigmentation region by PCR using primers IH and IY [2].

The described method is aimed only at identifying avirulent plague microbe strains lacking the chromosome region of pigmentation and cannot be used to evaluate the plasmid profile of Y. pestis strains.

There is also a method of identification and intraspecific differentiation of strains of the species Y. pestis by PCR using primers for three chromosome targets: “3a” 276 bp in size, “vlm12for / ISrev 216” 390 bp in size, “YP02258” 130 bp and to two targets of the plasmid pFra - “CDS39” of 418 bp, “traA” of 472 bp, and also to one target of Y. pseudotuberculosis “JS” - 223 bp [3].

This method allows to identify the affiliation of strains to the species Y. pestis and Y. pseudotuberculosis, to perform intraspecific differentiation of strains isolated in foci of Africa, Central Asia and the Caucasus, but is not intended to assess the virulence of the pathogen and determine its plasmid profile.

The literature describes a method for the diagnosis and identification of plague pathogen virulence markers using primers for caf1 gene fragments - 506 bp, lcrV - 800 bp, pla - 920 bp, irp2 - 300 bp in multilocus PCR with electrophoretic consideration of the results [4].

However, the authors themselves express uncertainty in the description of the high specificity and sensitivity of this method. In addition, for the detection of Yersinia pestis, it is necessary to use primers for species-specific DNA targets of chromosome localization, since the primers proposed by the authors in the study of plague microbe strains lacking both of these plasmids (and cases of isolation of such strains are known) will not allow differentiating the plague pathogen from other pathogenic Yersinia. Moreover, if the test sample contains Yersinia pseudotuberculosis or Yersinia enterocolitica DNA with a preserved island of high pathogenicity and pCad plasmid, a false-positive result will be obtained, which can lead to unreasonable anti-epidemic measures. Using the agarose gel electrophoresis method to take into account the results requires the allocation of an additional separate room in the PCR laboratory and the corresponding additional personnel, and the transfer of the PCR product to the electrophoresis gel increases the risk of DNA contamination, which can lead to a decrease in the quality of analysis.

A known set of polynucleotides specific for Yersinia pestis and a method for detecting Yersinia pestis, including 18 sequences of primers and probes, as well as 6 amplicons. Four of the target DNA used by the authors are located on the pFra plasmid, two on the chromosome [5].

This kit is intended only to identify the causative agent of plague, but not to determine the virulence and plasmid profile of the strains.

Also known are commercial diagnostic kits for detecting Yersinia pestis by real-time PCR from Invitrogen, PathAlert ™ Detection Kit for Yersinia pestis cat. No. BD1001 and Applied Biosystems, TaqMan Yersinia pestis detection kit, cat. No. 4382488. However, the kits are intended only for the detection of Yersinia pestis DNA without determining the properties of the pathogen.

Known commercial kit for detecting Yersinia pestis DNA "GenPest", manufactured by RosNIPCHI "Microbe". However, the use of this kit does not provide for the identification of isolated cultures and the determination of virulence and plasmid profile of strains.

A search by patents and scientific and technical sources of information showed the absence of a method and kit for the accelerated identification of Y. pestis strains with the possibility of studying a number of pathogen properties - virulence and plasmid profile.

The objective of the invention is the creation of a highly specific sensitive kit and method for the accelerated identification of plague microbe strains with the simultaneous differentiation of virulent and avirulent Y.pestis strains and determination of their plasmid profile by multilocus PCR with hybridization-fluorescence real-time results.

The technical result consists in increasing the efficiency of diagnostic studies due to the highly specific, rapid species identification of Y. pestis and differentiation of virulent and avirulent strains, as well as determining their plasmid profile.

The technical result is achieved by a kit for accelerated identification of the plague microbe with the simultaneous differentiation of virulent and avirulent strains of Y. pestis and the determination of their plasmid profile, including PCR mixture No. 1, containing primers SEQ ID N: 7, 8, 10, 11, 13, 14 and probes with different fluorescent labels at the 5′-end and a quencher at the 3′-end: SEQ ID N: 9 with the R6G tag and quencher RTQ1, SEQ ID N: 12 with the ROX tag and quencher BHQ2, SEQ ID N: 15 with the FAM tag and quencher RTQ1, PCR mixture 2 containing primers SEQ ID N: 16, 17, 19, 20, 22, 23 and probes: SEQ ID N: 18 labeled R6G and quencher RTQ1, SEQ ID N: 21 with me with FAM and RTQ1 quencher, SEQ ID N: 24 with a ROX tag and BHQ2 quencher with a primer / probe ratio of 2: 1 in each mixture. In addition, each of the mixtures contains sodium azide, four deoxynucleotide triphosphates. The kit also contains reagents for conducting multiplex PCR: TE buffer, Taq polymerase, a positive control sample and a mixture of reagents: magnesium chloride, 10-fold PCR buffer, deionized water.

The technical result is also achieved by the method of accelerated identification of the plague microbe with the simultaneous differentiation of virulent and avirulent strains of Y. pestis and the determination of their plasmid profile, in which a multiplex PCR mixture 1 and PCR mixture 2 from SEQ ID N primers are used to set up multiplex PCR: 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23 and probes SEQ ID N: 9, 12,15, 18, 21, 24 to the gene regions: 3a SEQ ID N: l , cafl SEQ ID N: 2, pla SEQ ID N: 3, irp2 SEQ ID N: 4, hmsH SEQ ID N: 5, lcrV SEQ ID N: 6, providing amplification of fragments of 147 bp, 138 bp ., 127 bp, 107 bp, 127 bp, 114 bp respectively, amplified according to the following parameters: 95 ° С - 15 min, 95 ° С - 30 s, 60 ° С - 30 s, 72 ° С - 30 s, repeat the cycle - 10 times / time, 95 ° С - 20 s, 56 ° С - 20 s - detection, 72 ° С -20 s, repeat the cycle - 35 times / time, with fluorescence measurement at 56 ° С (during the last 35 cycles) and taking into account the results on the FAM channel for the fluorescence label FAM, according to JOE channel for R6G label; ROX channel for ROX label; the identification of the pathogen, its differentiation and plasmid profile are evaluated by the increase in fluorescence signals in various combinations. The culture is evaluated as belonging to the species Y. pestis in the reaction with PCR mixture 1 through the channels FAM, JOE and ROX, or by FAM and JOE, or by JOE and ROX, or by JOE. The culture is evaluated as a virulent strain of Y. pestis in reaction with PCR mixture 2 through the channels FAM, JOE, ROX, or JOE and ROX. Plasmid pPst in the strain was detected in the reaction with PCR mixture 1 via one FAM channel, plasmid pFra through the ROX channel, and in the reaction with PCR mixture 2, the plasmid pCad was detected via the ROX channel.

When constructing the kit, particular importance was attached to the selection of DNA target fragments and, accordingly, primers for them. Since the creation of highly specific multiplex systems requires a combination of primers with certain characteristics (the absence of thermodynamically significant secondary structures) that do not cause a decrease in the efficiency of at least one of them when used together. The synthesis of oligonucleotides was carried out by the fofsfoamidite method, for example, on an ASM-800 DNA synthesizer from Bioset.

Justification for the selection of primers and probes

For the specific detection of Yersinia pestis DNA, many developers of gene-diagnostic preparations used genes of species-specific plasmids pFra (genes caf1, ymt) and pPst (pla, pst) [6, 7, 8, 9, 10, 11]. We chose caf1 and pla genes as markers of plasmids pFra and pPst, respectively, since the extensive international experience accumulated to date allows us to judge the high specificity and stability of these target DNAs.

However, for the detection of plague microbe strains lacking the plasmid pFra and / or pPst, the detection of fragments located on the chromosome of the plague pathogen is promising. One of such matrices can be a stable chromosome locus 41.7 kbp specific for Y. pestis. (NCBI GenBank No. AF350075), which according to L. Radnedge et al. (2001) delegated to other members of the genus Yersinia. The authors found that part of the indicated region, namely region 3a, is present in the genome of all the plague microbe strains studied by them, which was chosen as the chromosome localization target DNA for Y. pestis. However, data on the monomorphism of this site in the genome of plague microbe strains of various geographical origin, especially isolated on the territory of Russia, were absent.

Therefore, we sequenced a fragment of a 3a locus with a size of 522 bp 31 strains of the plague pathogen isolated on the territory of Russia and the CIS countries. It was found that the genomic polymorphism of this genetic element amounted to 0.4% with respect to the entire fragment (figure 1).

To select specific primers, homologous regions of the sequenced fragments of the 3a locus were used.

The pathogenicity of the plague microbe is determined by two main genetic determinants: the presence of a chromosomal island of high pathogenicity and the plasmid pCad, which carries the type III secretion system operon. The chromosome island of high pathogenicity of the plague pathogen contains genes of the iron assimilation system and consists of two regions: the ybt region, which synthesizes yersiniabactin, which chelates iron bound to eukaryotic proteins and transports it to the bacterial cell [12], and the hms region, which contains the operon hmsHFRS of the sorption system of hemin and exogenous dyes on the cell surface [13]. As a marker for the presence of the ybt region, we selected the irp2 gene, which is responsible for the synthesis of high molecular weight protein HMWP 2 (190 kDa). To detect the hms locus, the hmsH structural gene with high monomorphism was chosen as the target DNA.

One of the most commonly used markers for the presence of the plasmid pCad in Y. pestis strains is the V antigen or genes encoding it. V antigen (or LcrV protein) is a multifunctional determinant of pathogenicity that regulates the secretion of cytotoxic proteins from a bacterial cell into the cytosol of human or animal cells and has high protective properties [14]. Studies by T. C. Leal-Balbino et al. (2004) showed that pCad and, in particular, the lcrV gene are highly stable both in newly isolated strains of the plague microbe and in cultures subjected to long-term storage. Therefore, the lcrV gene was chosen as a marker of the calcium-dependent plasmid.

As a result of a thorough quality control of nucleotide sequence determination, the following consensus sequences of gene fragments were derived:

3a of SEQ ID No. 1 of 147 bp

caf1 SEQ ID NO: 138 bp

pla SEQ ID No. 3 of 127 bp

irp2 SEQ ID NO: 107 bp

hmsH SEQ ID No. 5 of 127 bp

lcrV SEQ ID NO: 114 bp

Based on the sequences of selected fragments of genes 3a (chromosome), caf1 (pFra tshazmid), pla (pPst plasmid), irp2 (island of high pathogenicity of the chromosomal pigmentation region), hmsH (hms locus of the chromosome pigmentation region), lcrV (pCad plasmid) the Primer Premier-V5 program (Premier Bio Soft International) and the BLAST algorithm selected oligonucleotide primers providing amplification of fragments of these loci of 147 bp, 138 bp, 127 bp, 107 bp, 127 bp b. 114 bp respectively. The primers were selected taking into account the possibility of their use in multiplex PCR with recording the results in the “real time” mode, and Taq Man hybridization probes were selected online on the website www.genscript.com.

Primers of SEQ ID No. 7 were selected for the region of the gene SEQ ID No. 1; SEQ ID No. 8 and probe SEQ ID No. 9, with a fluorescent label R6G and a fluorescence quencher RTQ1;

Primers of SEQ ID No. 10 were selected for the region of the gene SEQ ID No. 2; SEQ ID No. 11 and probe SEQ ID No. 12, with a fluorescence label ROX and a fluorescence quencher BHQ2;

Primers of SEQ ID No. 13 were selected for the region of the gene SEQ ID No. 3; SEQ ID No. 14 and probe SEQ ID No. 15, with a fluorescent label FAM and fluorescence quencher RTQ1;

Primers of SEQ ID No. 16 were selected for the gene region of SEQ ID No. 4; SEQ ID No. 17 and probe SEQ ID No. 18, with a fluorescent label R6G and a fluorescence quencher RTQ1;

Primers of SEQ ID No. 19 were selected for the gene region of SEQ ID No. 5; SEQ ID No. 20 and probe SEQ ID No. 21, with a fluorescent label FAM and fluorescence quencher RTQ1;

Primers of SEQ ID No. 22 were selected for the gene region of SEQ ID No. 6; SEQ ID No. 23 and probe SEQ ID No. 24, with a fluorescence label ROX and a fluorescence quencher BHQ2.

These sequences are presented in figure 2.

These primers and probes provide a high specificity of the reaction, do not form secondary structures (hairpins) and dimers, which contributes to their effective binding to matrix DNA. The optimal composition of the reaction mixture for performing multiplex PCR was experimentally established. In PCR mixtures No. 1 and 2, the selected concentrations of primers and probes were 2: 1, respectively, the amount of Taq polymerase enzyme at a concentration of 1.0 unit enough to ensure high specificity and efficiency of the reaction.

The mode of primer annealing and the effective number of amplification cycles were experimentally selected (Table 1).

Analytical Characteristics

A preliminary assessment of the analytical specificity of the used primers and probes using the on-line BLAST algorithm showed the lack of homology of the selected sequences with other microorganisms at a significance level of E = 10.

For the experimental assessment of the sensitivity and specificity of multiplex PCR with hybridization-fluorescence real-time results, we used quantitatively characterized dilutions of Y. pestis DNA at a concentration of 1 × 10 ³ -1 × 10 ⁷ m.k. / ml (table 2).

To characterize the specificity of PCR, a selection of strains from the target collection of various microorganisms was used. The data are presented in table 3.

As a result of the assessment, it was found that the selected primers and probes for identifying the plague microbe with the simultaneous differentiation of virulent and avirulent strains of Y. pestis and determination of their plasmid profile by the multiplex polymerase chain reaction with hybridization-fluorescence results are highly sensitive - 1 × 10 ⁴ m .k./ml and a specificity of 100%.

Based on these primers and probes, a kit was constructed that includes 9 tubes with screw caps with components: 1 microtube with PCR mixture No. 1, 1 microtube with PCR mixture No. 2, 2 microtube with a mixture of reagents for multiplex PCR, 1 microtube with Taq polymerase, 3 microtubes with TE buffer, 1 microtube with a positive control sample (FFP) Y. pestis DNA.

The multiplex PCR reagent mixture includes magnesium chloride, 10-fold PCR buffer, deionized water.

The PCR mixture No. 1 contains primers SEQ ID No. 7, 8, 10, 11, 13, 14 and probes SEQ ID No. 9,12,15, four deoxynucleotide triphosphate, sodium azide.

PCR mixture No. 2 contains primers SEQ ID No. 16, 17, 19, 20, 22, 23 and probes SEQ ID No. 18, 21, 24, four deoxynucleotide triphosphates, sodium azide.

The kit is intended for species identification of plague microbe strains, differentiation of virulent and avirulent Y. pestis strains, determination of their plasmid profile by multiplex polymerase chain reaction with hybridization-fluorescent results. The kit is designed for 50 determinations, including control samples. The kit has passed state tests and is being introduced into production.

The inventive method of accelerated identification of the plague microbe after DNA isolation by conventional methods includes the following steps:

a) conducting multiplex PCR;

b) evaluation of the results.

Carrying out PCR studies with a kit includes the simultaneous formulation of the reaction in two tubes for each test sample or control sample. In one tube, PCR mixture No. 1 is used, in the second PCR mixture No. 2. Taq polymerase and a mixture of reagents are used equally with both reaction mixtures.

PCR mixtures No. 1 and No. 2, a mixture of reagents, are extracted from the refrigerator; Taq polymerase, PF of Y. pestis DNA are removed from the freezer, completely thawed, mixed on a microcentrifuge / shaker and centrifuged at 3000-5000 rpm for 30 s to precipitate droplets from the walls of the tube.

To conduct the reaction in a separate tube of 0.6 ml or 1.5 ml, a reaction mixture is prepared containing: PCR mixture No. 1-5 μl, a mixture of reagents - 10 μl, Taq polymerase - 0.2 μl. The resulting mixture was thoroughly mixed and transferred to 15 μl in pre-prepared 0.2 ml tubes, the number of which corresponds to the number of test samples plus a positive and negative control sample. Similarly, prepare the reaction mixture for the reaction with PCR mixture No. 2.

Then, 10 μl of DNA from the studied samples is introduced into microtubes. 10 μl of TE buffer is added to the microtube for a negative control sample, and 10 μl of Y. pestis DNA PCO for a positive control sample. All tubes are placed in a Rotor Gene thermal cycler.

For work with the Rotor Gene 3000 device use the Rotor Gene 6 program, with the device. "Rotor Gene" 6000 - program "Rotor Gene 6000" version 1.7 (build 67) or higher.

The registration of the reaction results using PCR mixture No. 1 and PCR mixture No. 2 is carried out uniformly.

The results are taken into account based on the presence (or absence) of the intersection of the fluorescence curve with the threshold line set at the appropriate level, and the digital value of the threshold cycle “Ct” is indicated in the corresponding column of the results table on the instrument panel.

The threshold Ct value for positive samples on the FAM, JOE, ROX channels is no more than 33. If the value is received no more than 33, the result is considered positive.

Evaluation of the results.

Analysis of the results obtained with PCR mixture No. 1 and PCR mixture No. 2 is performed sequentially. In the case of positive results with PCR mixture No. 1, the results obtained with PCR mixture No. 2 are taken into account. In the case of a negative result with PCR mixture No. 1, the results with PCR mixture No. 2 are not taken into account.

PCR time is 2 hours.

Accounting and evaluation of the results allows you to:

- determine the species affiliation of the studied cultures to Y. pestis based on the registration of signals through the channels FAM, JOE, ROX with PCR mixture No. 1 according to evaluation table 4;

- to differentiate virulent and avirulent strains of Y. pestis based on the registration of signals through the channels FAM, JOE, ROX with PCR mixture No. 2 according to evaluation table 5;

- determine the plasmid profile of Y. pestis strains based on the registration of signals on the FAM and ROX channels with PCR mixture No. 1 and the ROX channel with PCR mixture No. 2 according to evaluation table 6.

Example

The material for the study was 5 encrypted samples, in which the causative agent of the plague and representatives of a different kind were present.

The preparation of samples of bacterial suspensions was performed in accordance with the requirements of SP 13.1285-03 “Safety of work with microorganisms of pathogenicity groups I-II” (Moscow, 2003).

The disinfection of the studied bacterial suspensions was carried out in accordance with MU 1.3.2569-09 “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of I-IV pathogenicity groups” (Moscow, 2009).

DNA isolation was performed according to the generally accepted scheme using the registered commercial Ribo-sorb kit, in accordance with the instructions for the kit.

Multiplex PCR was carried out simultaneously in two tubes for each test sample or control sample. PCR mixture No. 1, PCR mixture No. 2, a mixture of reagents were extracted from the refrigerator, and Taq polymerase, Y. pestis PKO were completely thawed from the freezer, mixed on a microcentrifuge / shaker and centrifuged at 3000-5000 rpm 30 s to precipitate droplets from the walls of the tube.

To conduct the reaction with PCR mixture No. 1, 6 microtubes with a volume of 0.2 ml were selected, corresponding to the number of test samples for negative control of DNA extraction (OKB), and another 2 for positive and negative controls. In the same way, microtubes were selected for the reaction with PCR mixture No. 2.

In a separate 0.6 ml microtube, the reaction mixture was prepared from the calculation: PCR mixture No. 1 - 40 μl, PCR mixture No. 2 - 80 μl, Taq polymerase - 1.6 μl. The prepared mixture was thoroughly mixed and transferred to 15 μl in 6 prepared tubes with a volume of 0.2 ml. Similarly, the reaction mixture was prepared for the reaction with PCR mixture No. 2.

Then, in 10 test tubes with PCR mixture No. 1, 10 μl of DNA from the studied samples was added. 10 μl of TE buffer was added to a microtube with a negative amplification control (K-), and 10 μl of Y. pestis DNA and OKV DNA were added to a microtube with a positive amplification control (K +). Similarly, the reaction was carried out with PCR mixture No. 2. Prepared microtubes were placed in a RotorGene thermal cycler.

The following amplification program was set:

1) Hold temperature - 95 ° C - 5 min 2) Cycling / Cycling - 95 ° C - 30 sec 60 ° C - 30 sec 72 ° C - 30 sec

Cycle repeats - Repeat 10 times / time.

3) Susling2 / Cycle2 95 ° C - 20 sec 56 ° C - 20 sec. - Detection 72 ° C - 20 sec

Cycle repeats / cycle repeat - 35 times / time.

Fluorescence was measured at 56 ° C (in the second cycling unit) using the channels FAM / Green, JOE / Yellow, ROX / Orange.

The calibration parameters were set: for the FAM / Green, JOE / Yellow and ROX / Orange channels, they are indicated in the column Min Reading. The signal value is 5, and in the column Max Reading / Maxim. Signal value 10.

The registration of the reaction results using PCR mixture No. 1 and PCR mixture No. 2 was carried out uniformly.

Registration of amplification results via the FAM / Green channel

1. Activated the modes "Dynamic tube" / "Dynamic.fon", "Slope Correct" / "Correction. bias ”,“ Ignore First - 10 Cycles ”/“ Ignore. the first - 10 cycles. "

2. Set the parameters “Outlier Removal” / “Elimination of emissions” - 30%.

3. Set the threshold line value = 0.05, activate the option “Eliminate Cycles Before - 8” / “Exclude cycles to - 8”.

4. In the table of results fixed values of the threshold cycle (Ct) for each sample.

Registration of amplification results via the JOE / Yellow channel

1. Activated the "Dynamic tube" / "Dynamic. background, Slope Correct. bias ”,“ Ignore First - 10 Cycles ”/“ Ignore. the first - 10 cycles. "

2. Set the parameters “Outlier Removal” / “Elimination of emissions” - 15%.

3. Set the threshold line value = 0.05, activate the option “Eliminate Cycles Before - 8” / “Exclude cycles to - 8”.

4. In the table of results fixed values of the threshold cycle (Ct) for each sample.

Registration of amplification results via ROX / Orange

1. Activated the "Dynamic tube" / "Dynamic. background, Slope Correct. bias ”,“ Ignore First - 10 Cycles ”/“ Ignore. the first - 10 cycles. "

2. Set the parameters “Outlier Removal” / “Elimination of emissions” - 15%.

3. Set the threshold line value = 0.05, activate the option “Eliminate Cycles Before - 8” / “Exclude cycles to - 8”.

4. In the table of results fixed values of the threshold cycle (Ct) for each sample.

The results of PCR are presented in the figures and tables. The determination of the species affiliation of the studied cultures based on the registration of signals through the channels FAM, JOE and ROX with PCR mixture No. 1 are shown in FIGS. 3-5 and table 7.

Based on the results, it was concluded that sample No. 1 did not contain a culture belonging to the species Y. pestis (negative result), and the results obtained in PCR with this sample were not subject to further accounting with PCR mixture No. 2.

The strains present in samples No. 2, 3, 4, 5 belong to the species Y. pestis (positive result), and results will be recorded for them with PCR mixture No. 2.

Differentiation of virulent plague microbe strains from avirulent strains in samples Nos. 2, 3, 4, 5 was carried out on the basis of registration of signals through FAM, JOE and ROX channels with PCR mixture No. 2 (Figs. 6-8, Table 8).

Based on the results, it was concluded that the plague microbe strains present in samples 2 and 3 are virulent, while samples 3 and 4 contain avirulent Y. pestis strains.

The determination of the plasmid profile of Y. pestis strains was carried out based on the registration of signals on the FAM and ROX channels with PCR mixture No. 1 and the ROX channel with PCR mixture No. 2 (table 9).

The data obtained showed that the strain Y. pestis in sample No. 2 contains plasmids pPst, pFra, pCad; the strain in sample No. 3 contains the plasmids pFra and pCad, but lacks the plasmid pPst; the strain in sample No. 4 contains the plasmid pPst, but lacks the plasmids pFra and pCad; the strain in sample No. 5 contains plasmids pPst, pFra, pCad.

The final conclusion on the results of multiplex PCR analysis of 5 encrypted samples using the developed set and method

Sample No. 1 - does not contain Y. pestis.

Sample No. 2 - contains a virulent strain of Y. pestis with plasmids pPst, pFra, pCad.

Sample No. 3 - contains a virulent strain of Y. pestis with plasmids pFra and pCad, but lacks the plasmid pPst.

Sample No. 4 - contains an avirulent strain of Y. pestis with the plasmid pPst, but lacks the plasmids pFra and pCad.

Sample No. 5 - contains an avirulent strain of Y. pestis with plasmids pPst, pFra, pCad.

After deciphering the samples, the results obtained in all cases coincided with the characteristics of the strains used to prepare the encrypted samples when studying them in bacteriological, serological and biological methods. The strains were obtained from the State collection of pathogenic bacteria FGUZ RosNIPCHI "Microbe" (table 10).

Thus, the claimed group of inventions has novelty. Target DNAs were selected — sequences of gene fragments 3a of SEQ ID NO: 147 bp, responsible for species specificity; caf1 SEQ ID NO: 138 bp (plasmid pFra); pla SEQ ID No. 3 of 127 bp (plasmid pPst); irp2 SEQ ID NO: 107 bp; hmsH SEQ ID NO: 127 bp; lcrV SEQ ID NO: 114 bp Primers and hybridization probes were selected, the optimal composition of the reaction mixtures for performing real-time multiplex PCR was determined, and the optimal reaction conditions, accounting and evaluation features, which made it possible to identify the culture, determine its virulence and plasmid profile, and also achieve a high level sensitivity and specificity, reduce analysis time. The kit is at the stage of introduction into production - it has passed preliminary, technical and medical tests. An additional examination and registration is underway at Roszdravnadzor.

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Table 1 Primer Annealing Temperature The presence of homogeneous amplification channels FAM, JOE and ROX PCR mixture No. 1 PCR mixture No. 2 62 ° C - - 60 ° C - - 58 ° C ± ± 56 ° C + + 54 ° C ± ± 52 ° C - - 50 ° C - - Amplification cycles Effective amplification of Yersinia pestis DNA at a concentration of 1 × 10 ⁴ MK./ml PCR mixture No. 1 PCR mixture No. 2 25 - - thirty ± ± 35 + + 40 + +

table 2 The concentration of Y. pestis in the sample Amplification via FAM, JOE and ROX channels PCR mixture No. 1 PCR mixture No. 2 1 × 10 ² m.k. / ml - - 5 × 10 ² m.k./ml - - 1 × 10 ³ m.k. / ml - - 5 × 10 ³ m.k. / ml ± ± 1 × 10 ⁴ m.k. / ml + + 5 × 10 ⁴ m.k./ml + + 1 × 10 ⁵ m.k./ml + +

Table 3 Type of microorganism The number of strains Amplification via FAM, JOE and ROX channels PCR mixture No. 1 PCR mixture No. 2 Y.pestis 31 + + Y.pseudotuberculosis 2 - - Y.enterocolitica 2 - - E.coli 2 - -

Table 4 PCR mixture PCR mixture No. 1 results Fluorescence channel Fam Joe Rox Genetic marker pla (plasmid pPst) 3a (chromosome) caf1 (plasmid pFra) Samples Threshold Cycle Values (Ct) OKV, K- Lack of fluorescence Lack of fluorescence Lack of fluorescence Analysis results are subject to accounting. The presence of fluorescence in at least one of the channels Analysis results are not subject to accounting K + ≤33 ≤33 ≤33 Analysis results are subject to accounting. Absence of fluorescence in at least one of the channels Analysis results are not subject to accounting Test samples ≤33 ≤33 ≤33 The culture belongs to the species Yersinia pestis ≤33 ≤33 Lack of fluorescence Lack of fluorescence ≤33 ≤33 Lack of fluorescence ≤33 Lack of fluorescence Lack of fluorescence Lack of fluorescence Lack of fluorescence The culture does not belong to the species Yersinia pestis

Table 5 PCR mixture PCR mixture No. 2 results Fluorescence channel Fam Joe Rox Genetic marker hmsH (chromosomal pigmentation region) irp2 (chromosomal pigmentation region) lcrM (plasmid pCad) Samples Threshold Cycle Values (Ct) OKV, K- Lack of fluorescence Lack of fluorescence Lack of fluorescence Analysis results are subject to accounting. The presence of fluorescence in at least one of the channels Analysis results are not subject to accounting FFP DNA Y.pestis ≤33 ≤33 ≤33 Analysis results are subject to accounting. Absence of fluorescence in at least one of the channels Analysis results are not subject to accounting Test samples ≤33 ≤33 Lack of fluorescence The strain of Yersinia pestis is avirulent ≤33 Lack of fluorescence ≤33 ≤33 Lack of fluorescence Lack of fluorescence Lack of fluorescence ≤33 Lack of fluorescence Lack of fluorescence Lack of fluorescence ≤33 Lack of fluorescence Lack of fluorescence Lack of fluorescence ≤33 ≤33 ≤33 The strain of Yersinia pestis is virulent Lack of fluorescence ≤33 ≤33

Table 6 PCR mixture PCR mixture No. 1 PCR No. 2 results Fluorescence channel Fam Rox Rox Genetic marker pla caf1 lcrV Samples Threshold Cycle Values (Ct) OKV, K- Lack of fluorescence Lack of fluorescence Lack of fluorescence Analysis results are subject to accounting. The presence of fluorescence in at least one of the channels Analysis results are not subject to accounting K + ≤33 ≤33 ≤33 Analysis results are subject to accounting. Absence of fluorescence in at least one of the channels Analysis results are not subject to accounting Test samples ≤33 ≤33 ≤33 The strain contains plasmids pCad, pPst, pFra ≤33 Lack of fluorescence ≤33 The strain contains plasmids pCad, pPst Lack of fluorescence ≤33 ≤33 The strain contains plasmids pCad, pFra ≤33 ≤33 Lack of fluorescence The strain contains plasmids pPst, pFra Lack of fluorescence Lack of fluorescence ≤33 The strain contains the plasmid pCad ≤33 Lack of fluorescence Lack of fluorescence The strain contains the plasmid pPst Lack of fluorescence ≤33 Lack of fluorescence The strain contains plasmid pFra Lack of fluorescence Lack of fluorescence Lack of fluorescence The strain does not contain plasmids pCad, pPst, pFra

Table 7 Fluorescence channel Fam Joe Rox results Genetic marker pla (plasmid pPst) 3a (chromosome) caf1 (plasmid pFra) Samples Threshold Cycle Values (Ct) Sample No. 1 Lack of fluorescence Lack of fluorescence Lack of fluorescence The culture does not belong to the species Y. pestis Sample No. 2 17.94 17.78 15.06 Culture belongs to the species Y. pestis Sample No. 3 Lack of fluorescence 20.33 20.58 Culture belongs to the species Y. pestis Sample No. 4 18.88 20.15 Lack of fluorescence Culture belongs to the species Y. pestis Sample No. 5 22.24 22.32 19.38 Culture belongs to the species Y. pestis

Table 8 Fluorescence channel Fam Joe Rox results Genetic marker hmsH (chromosomal pigmentation region) irp2 (chromosomal pigmentation region) lcrV (plasmid pCad) Samples Threshold Cycle Values (Ct) Sample No. 2 19.19 20.39 16.04 Strain Y.pestis virulent Sample No. 3 21.79 23.16 19.36 Strain Y.pestis virulent Sample No. 4 18.71 19.39 Lack of fluorescence Strain Y.pestis avirulent Sample No. 5 Lack of fluorescence Lack of fluorescence 18.64 Strain Y.pestis avirulent

Table 9 PCR mixture No. 1 PCR mixture No. 2 results Fluorescence channel Fam Rox Rox Genetic marker pla (plasmid pPst) caf1 (plasmid pFra) lcrV (plasmid pCad) Samples Threshold Cycle Values (Ct) Sample No. 2 17.94 15.06 16.04 Strain Y. pestis contains plasmids pPst, pFra, pCad Sample No. 3 Lack of fluorescence 20.58 19.36 Strain Y. pestis contains plasmids pFra, pCad Sample No. 4 18.88 Lack of fluorescence Lack of fluorescence Strain Y.pestis contains the plasmid pPst Sample No. 5 22.24 19.38 18.64 Strain Y. pestis contains plasmids pPst, pFra, pCad

Table 10 Samples Sample strains Biological determination of virulence Determination of plasmids by biochemical method Plasmid pFra Plasmid pPst Plasmid pCad Sample No. 1 Yersinia pseudotuberculosis 312 at a concentration of 1 × 10 ⁴ mk./ml n / a - - - Sample No. 2 Yersinia pestis C-624 at a concentration of 1 × 10 ⁴ m.k. / ml virulent + + + Sample No. 3 Yersinia pestis 6499 at a concentration of 1 × 104 m.k. / ml virulent + - + Sample No. 4 Yersinia pestis 165 at a concentration of 1 × 10 ⁴ m.k. / ml avirulent - + - Sample No. 5 Yersinia pestis EV NIIEG at a concentration of 1 × 10 ⁴ m.k. / ml avirulent + + +

Claims (2)

1. A kit for accelerated identification of plague microbe strains with simultaneous differentiation of virulent and avirulent Y.pestis strains and determination of their plasmid profile, including N: 1 PCR mixture containing primers SEQ ID N: 7, 8, 10, 11, 13, 14 and probes with different fluorescent labels at the 5′-end and quenchers at the 3′-end: SEQ ID N: 9 labeled R6G and quencher RTQ1, SEQ ID N: 12 labeled ROX and quencher BHQ2, SEQ ID N: 15 tagged FAM and quencher RTQ1, PCR mixture N: 2 containing primers SEQ ID N: 16, 17, 19, 20, 22, 23 and probes SEQ ID N: 18 with the label R6G and quencher RTQ1, SEQ ID N: 21 with the label FAM and quencher RTQ1, SEQ ID N: 24 with a ROX label and a BHQ2 quencher with a primer / probe ratio of 2: 1 in each mixture. 2. The method of accelerated identification of strains of the plague microbe with the simultaneous differentiation of virulent and avirulent strains of Y. pestis and the determination of their plasmid profile, characterized in that they carry out multiplex PCR in two tubes in one step using the kit according to claim 1, containing N PCR mixture : 1, including primers SEQ ID N: 7, 8, 10, 11, 13, 14 and probes SEQ ID N: 9, 12, 15, and PCR mixture N: 2, including primers SEQ ID N: 16, 17, 19, 20, 22, 23 and probes SEQ ID N: 18, 21, 24, to gene regions 3a SEQ ID N: 1 - 147 bp, cafl SEQ ID N: 2 - 138 bp, pla SEQ ID N: 3 - 127 bp, irp2 SEQ ID N: 4 - 107 bp, hmsH SEQ ID N: 5 - 127 bp, IcrV SEQ ID N: 6 - 114 bp, providing amplification of fragments of the required size; primers are annealed at a temperature of 60 ° C for 10 cycles, then at a temperature of 56 ° C for 35 cycles; identify Y. pestis when registering signals from the labels of all probes of the PCR mixture N: 1 or by a combination of signals from SEQ ID N: 9 and SEQ ID N: 15, SEQ ID N: 9 and SEQ ID N: 12, or by the signal from SEQ ID N: 9; the virulent strain of Y. pestis is identified when registering fluorescent signals from the labels of all probes of the PCR mixture N: 2 or by a combination of signals from SEQ ID N: 18 and SEQ ID N: 24; the plasmid profile of the strain Y. pestis is determined based on the registration of the fluorescent signal from the probe label SEQ ID N: 24 PCR mixture N: 2 in the presence of plasmid pCad, based on the registration of the fluorescent signal from the probe label SEQ ID N: 15 PCR mixture N: 1 in the presence of plasmid pPst, based on the registration of the fluorescent signal from the probe label SEQ ID N: 12 PCR mixture N: 1 in the presence of plasmid pFra.

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