RU2703803C1 - SET OF OLIGONUCLEOTIDE PRIMERS Ft 101 AND A METHOD FOR DETERMINING FRANCISELLA TULARENSIS (VERSIONS) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, molecular biology and medical microbiology. A set of oligonucleotide primers for the loop isothermal amplification reaction (LAMP), which enables to amplify bacteria-specific Francisella tularensis fragments of DNA from the target gene having the nucleotide sequence SEQ ID No. 1. For amplification of specific DNA fragments of Francisella tularensis in the loop isothermal amplification reaction, a set of oligonucleotide primers is proposed, which includes: a FIP primer – "acpFt101-F3", having a nucleotide sequence SEQ ID No. 2, primer BIP – "acpFt101-B3", having nucleotide sequence SEQ ID No. 3, primer F3 – "acpFt101-FIP", having nucleotide sequence SEQ ID No. 4, primer B3 – "acpFt101-BIP" having nucleotide sequence SEQ ID No. 5.

EFFECT: declared invention allows detecting presence of tularemia pathogen – Francisella tularensis within 1–2 hours with high specificity in samples of pure cultures in concentrations from 10 to 1,000 m.c. in ml (depending on strains).

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Description

The invention relates to medicine, biotechnology, molecular biology and can be used for laboratory diagnosis of tularemia, both in practical health care and for research in the field of microbiology, epidemiology and pathogenesis of tularemia.

Tularemia is a zoonotic natural focal infection, the etiological agent of which is Francisella tularensis, a gram-negative intracellular bacterium that regularly causes epidemic outbreaks in animals and humans.

The species F. tularensis has four subspecies: holarctica, mediasiatica, novicida, and tularensis. Of particular danger is F. tularensis subsp. tularensis. It has been found that inhaling 10 bacteria can cause disease in humans (McCrumb F.R. 1961. Aerosol infection of man with Pasteurella tularensis. Bacteriological Reviews 25: 262-267). Due to the high infectivity in the airborne pathway of infection, F. tularensis is classified as a hazard level 1 bioagent (category A).

Despite the fact that the "gold" standard "for diagnosing tularemia is still the cultivation of a microorganism, it is usually not carried out due to the low sensitivity and exactingness to the working conditions. For the detection and identification of F. tularensis, methods such as biosensors, cultivation, immunochromatographic analysis, PCR, biochips on a liquid or solid basis, analytical chemistry methods (gas chromatography, mass spectrometry, etc.) have been tried. Among these methods for detecting F. tularensis, PCR is dominant. When monitoring the natural foci of tularemia and laboratory testing of the material for the presence of F. tularensis only using PCR, it is possible to identify single bacteria of the causative agent of tularemia (highly sensitive and highly specific nested variant) (Tatarnikov S.A., Mazepa A.V., Balakhonov S.V. Optimization of the Nested-variant of the polymerase chain reaction for monitoring the natural foci of tularemia. Infection and immunity. 2013. V. 3. No. 2. P. 175).

Existing commercial test systems and experimental samples of diagnostic preparations make it possible to characterize strains for the presence of genetic determinants specific for the species F. tularensis, various subspecies (chilf, RD1 genes, etc.), strains of different virulence (pdpA, pdpD), etc. (Laboratory Diagnosis of Dangerous Infectious Diseases: A Practical Guide. Edited by G.G. Onishchenko, V.V. Kutyreva. - M .: ZAO "Shiko", 2013. - 560 p., Tatarnikov S.A., Mazepa A.V. ., Dubrovina VI, Voitkova VV Identification of the pdpD and pdpA genomic regions in the tularemia microbe of different subspecies. In the book “Biological safety in the modern world.” 2009. P. 69-71).

Studies of molecular genetic amplification technology - loop-mediated isothermal amplification (LAMP) - showed high specificity and amplification efficiency, which is achieved under isothermal reaction conditions (Kaneko H., Kawana T., Fukushima E., Suzutani T. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J. Biochem. Biophys. Methods. 2007. 70 (3): 499-501; Soto E., Hawke JP, Fernandez D., Morales JA Francisella sp., an emerging pathogen of tilapia, Oreochromis niloticus (L.), in Costa Rica. J Fish Dis. 2009; 32 (8): 713-22). LAMP uses 4 primers that are homologous to six regions of the target gene. The reaction proceeds at a constant temperature using an enzyme that catalyzes the synthesis of new chains with a shift in the old ones. Amplification and detection of a gene can be carried out in one step by incubating a mixture of samples, primers, DNA polymerase with the function of displacing chains and substrates at a constant temperature (about 65 ° C). The amplification efficiency is very high, within 15-60 minutes the DNA is amplified 10 ⁹ -10 ¹⁰ times. Due to its high specificity, the presence of the amplified product clearly indicates the presence of the target gene.

It is known that a similar method is used to detect the presence of Francisella piscicida, an etiological agent of Atlantic cod francicellosis (Gadus morhua) (Caipang S.M., Kulkarni A., Brinchmann MF, Korsnes K., Kiron V. Detection of Francisella piscicida in Atlantic cod (Gadus morhua L) by the loop-mediated isothermal amplification (LAMP) reaction. Vet J 2010; 184 (3): 357-61). To detect the pathogen GroEL gene, a set of primers was developed (two external and two internal). The selection of LAMP conditions showed an optimal reaction temperature of 63 ° C for 1 h using bacterial genomic DNA as a template. The reaction products were visualized in ultraviolet light and analyzed by agarose gel electrophoresis. The method turned out to be highly specific for the detection of F. piscicida and is 100 times more sensitive than conventional PCR. The use of LAMP analysis is possible not only for DNA strains, but also for Atlantic cod organ suspensions. However, for pathogenic F. tularensis species for humans, information on the developed primers and (or) diagnostic LAMP tests was not found in open sources.

A known enzyme for loop isothermal amplification is Bst polymerase isolated from Bacillus stearothermophilus (Nagamine K., Watanabe K., Ohtsuka K., Hase T., Notomi T., 2001. Loop-mediated isothermal amplification reaction using a non-denatured template. Clin. Chem., 47: 1742-1743). Bst polymerase is active at temperatures up to 66 ° C, but the optimum temperature for it is 60 ° C. The success of isothermal amplification depends on the activity of the enzymes to displace the chain and on their ability to pass through such complex regions of DNA as hairpins.

However, in scientific publications there are reports of the appearance of false positive signals when working with Bst polymerase.

A search of patent databases and scientific and technical sources of information showed a lack of information about domestic kits designed to detect F. tularensis by loop isothermal amplification. Therefore, there is a need to create such a diagnosticum for fast, sensitive and specific detection of tularemia microbe DNA.

The technical result of the invention is the development of highly specific oligonucleotide primers for a quick, specific and sensitive method for the detection of bacteria of the species F. tularensis in biological samples based on loop isothermal amplification of a fragment of the target gene.

The technical result is achieved by the fact that a set of original primers is proposed SEQ ID No: 2 (direct external primer acpFt101-F3 F. tularensis);

SEQ ID No: 3 (reverse external primer acpFt101-B3 F. tularensis);

SEQ ID No: 4 (direct internal primer acpFt101-FIP F. tularensis);

SEQ ID No: 5 (reverse internal primer acpFt101-BIP F. tularensis) for the detection of tularemia microbe using the SD polymerase enzyme (Ignatov K.V., Barsova EV, Fradkov AF, Blagodatskich KA, Kramarova TV, Kramarov VM A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification. BioTechniques. 2014; 57: 81-7) in a loop isothermal amplification reaction under the following conditions: preliminary heating of the mixture 92 ° C for 2 min, amplification 60 ° C - 60 min, the detection of the amplification product is carried out using agarose gel electrophoresis using a characteristic electrophoregram (varia NT 1) or staining the products of the amplification reaction using the intercalating dye SYBR Green I (option 2)

The technical result is also achieved through the use of the target gene as acA encoding acid phosphatase A.

Justification of the choice of oligonucleotide primers and target DNA.

Sequences of six acid phosphatases (acPA, acpB, acpC acpD, hapA, and hapB) were found in the genome of the tularemia microbe. They inhibit the oxidative burst in the phagosomes of infected macrophages and thereby contribute to the survival of the microbe in the host cells. Gene defects or their loss reduces the virulent properties of the microbe (Felts RL, Reilly TJ, Tanner JJ Crystallization of AcpA, a respiratory burst-inhibiting acid phosphatase from Francisella tularensis. Biochim. Biophys. Acta. 2005. Aug 31; 1752 (1): 107 -10). The selected asA gene encoding acid phosphatase A is present in the genome of the virulent for humans subspecies F. tularensis. The sequence of SEQ ID No. 1 (target gene acPA) for the selection of primers for the detection of DNA of the causative agent of tularemia by loop isothermal amplification was obtained from the GenBank NCBI database (National Center for Biotechnology Information, USA).

A strain of F. tularensis subsp. Was used as a positive control in the experiments. holarctica 15 NIIEG, using for the isolation of DNA disinfected suspensions of the microorganism in concentrations from 1 × 10 ⁶ MK./ml to 1 × 10 ¹ MK./ml. The primers were tested on a set of strains of tularemia pathogens of the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk."

The sensitivity of the amplification reaction with primers was evaluated by studying DNA samples isolated from ten-fold dilutions of the strain F. tularensis subsp. holarctica 15 NIIEG.

The invention is illustrated by the following graphic materials (table. 1, Fig. 1-4) and examples (1-3).

Table 1. Characterization of the selected oligonucleotide primers for the detection of the causative agent of tularemia.

FIG. 1. The sequence of SEQ ID No. 1 is the nucleotide sequence of the ACPA gene from the genome of the strain Francisella tularensis subsp. tularensis SCHU S4. The gray rectangles indicate the primer annealing sites and the sequences complementary to them.

Fig 2. Determination of the specificity of LAMP primers SEQ ID No. 2; SEQ ID No. 3, SEQ ID No. 4; SEQ ID No. 5 for the detection of F. tularensis using SD polymerase. Designations: 1 - DNA of F. tularensis subsp. holarctica 15 NIIEG; 2 - DNA of F. tularensis subsp. holarctica 503; 3 - DNA of F. tularensis subsp. tularensis Schu S4; 4 - DNA of F. tularensis subsp. mediasiatica 120; 5 - DNA of F. tularensis subsp. mediasiatica A678; 6 - F. tularensis subsp. mediasiatica A198; 7 - DNA of B. anthracis STI-1; 8 - DNA of Y. pestis subsp. pestis bv. antiqua I3449; 9 - DNA of Y. pseudotuberculosis C79; 10 - DNA of V. cholerae Eltor M878; 11 - DNA of Escherichia coli JM83; 12 - B. suis 1330 DNA; 13 - DNA of B. abortus 19, 14 - DNA of B. melitensis 16-M, 15 - DNA of L. pneumophila subsp. pneumophila serogroup 1, ATCC 33152; 16 - DNA of L. pneumophila subsp. pneumophila serogroup 6, ATCC 33215; 17 - negative control LAMP (water); 18 - molecular weight marker (100-1000 bp).

Fig 3 The results of determining the sensitivity of the set of LAMP primers SEQ ID No. 2; SEQ ID No. 3, SEQ ID No. 4; SEQ ID No. 5 using SD polymerase. Used DNA from the vaccine strain F. tularensis subsp. holarctica 15 NIIEG.

Designations: 1 - DNA from 1 × 10 ⁶ m.k. 2 - DNA from 1 × 10 ⁵ mk; 3 - DNA from 1 × 10 ⁴ m.k. 4 - DNA from 1 × 10 ³ m.k. 5 - DNA from 1 × 10 ² mk; 6 - DNA from 1 × 10 ¹ MK; 7 - DNA from 1 m.k .; 8 - negative control of PCR (water); 9 - molecular weight marker (100-1000 bp).

FIG. 4. The results of determining the sensitivity of the set of LAMP primers SEQ ID No. 2; SEQ ID No. 3, SEQ ID No. 4; SEQ ID No. 5 using SD polymerase and staining the reaction products of SYBR Green I. DNA was used from the vaccine strain F. tularensis subsp. holarctica 15 NIIEG.

Designations: 1 - DNA from 1 × 10 ⁶ m.k. 2 - DNA from 1 × 10 ⁵ mk; 3 - DNA from 1 × 10 ⁴ m.k. 4 - DNA from 1 × 10 ³ m.k. 5 - DNA from 1 × 10 ² mk; 6 - DNA of 1 × ¹⁰ January MK .; 7 - DNA from 1 m.k .; 8 - negative control of PCR (water).

Examples of specific performance.

Example 1. The method of designing oligonucleotide primers for the detection of DNA of tularemia pathogens by loop isothermal amplification.

Based on the in silico analysis of the nucleotide sequences of the tularemia pathogen present in the Genbank database, a 1545 bp genome fragment was selected for the construction of direct and reverse external primers F3 and B3, direct and reverse internal primers FTP and BIP. (GenBank NCBI, GenelD: 3192410 ). encoding acid phosphatase A. To the site of the gene SEQ ID No. 1 (Fig. 1) primers selected SEQ ID No. 2; SEQ ID No. 3, SEQ ID No. 4; SEQ ID No. 5. The estimated length of the proposed amplicon flanked by primers SEQ ID No. 4; SEQ ID No. 5, amounted to 154 bp (tab. 1). In FIG. 1 shows primer annealing sites and sequences complementary to them, which are indicated by gray rectangles.

When choosing oligonucleotide primers, the Primer Explorer 5 program for calculating primers on-line was used (http://primerexplorer.jp/lampv5e/index.html). When selecting primers were guided by the requirements for the oligonucleotides used in LAMP. The formation of secondary structures (dimers, hairpins) was analyzed using the selected primers using the mfold computer program (http://unafold.rna.albany.edu/?q=mfold/DNA-Folding-Form). Structures of all

oligonucleotides were compared using the NCBI BLAST information resource (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with DNA sequences hosted in the GenBank database. When conducting this analysis, oligonucleotides with significant homology with the DNA of any other organisms were excluded. To determine the specificity of the primers, in silico PCR was simulated for sequenced genomes of microorganisms using In silico PCR amplification (http://insilico.ehu.es/PCR/). The theoretical suitability of the primers for the successful initiation of the amplification and hybridization reactions is shown.

Example 2. Amplification of specific DNA fragments using the designed primers for the detection of the causative agent of tularemia by loop isothermal amplification.

A 50 μl reaction mixture contained 1 × SD buffer (RusEnzyme, Russia), 40 units. SD polymerases (RusEnzyme, Russia), 3.5 mM MgCl ₂ , 0.5 mM each dNTP, 4 primers: 0.2 μM SEQ ID No. 2 (acpFt101F3), 0.2 μM SEQ ID No. 3 (acpFt101B3), 0.8 μM SEQ ID NO: 4 (acpFt101BIP), 0.8 μM SEQ ID NO: 5 (acpFt101FIP), 5 μl matrix. The reaction was carried out at a temperature of 60 ° C for 60 min with preliminary heating at a temperature of 92 ° C for 2 min on a Tertsik thermocycler (DNA-Technology). Detection of LAMP products was carried out by electrophoresis in 1.2% agarose gel in TAE buffer. (Variant 1.) Or the LAMP products were detected by staining the amplifications with the SYBR Green I intercalating dye. The samples were painted immediately after the reaction by adding dye to the reaction to a final concentration of 10 ×. (Option 2). In both variants of detection, visualization was carried out on a UV transilluminator at a wavelength of 312 nm.

The amplification result of each strain of F. tularensis was considered positive in the case of the appearance of strips in the form of a ladder (option 1) or the appearance of a bright green color after adding SYBR Green I to the amplifiers (option 2). Fig 2 displays the electrophoregram obtained by amplification of DNA strains of the causative agent of tularemia with a set of primers selected for the sequence of SEQ ID No. 1.

Example 3. Determination of the sensitivity and specificity of the amplification reaction using the developed oligonucleotide primers to the sequence SEQ ID No. 1 for the identification of DNA of the causative agent of tularemia.

The sensitivity of the amplification reaction with the developed specific primers SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 was evaluated in the study of DNA samples isolated from bacterial suspensions of F. tularensis cells. Francisella strains were grown on FT-agar Petri dishes supplemented with polymyxin B (100 μg / ml) for 48 h at a temperature of (37 + 1) ° С. Bacterial cell suspensions were prepared in 2 ml of 0.9% sterile sodium chloride solution according to the industry standard sample of turbidity of 10 units of GISK named after L.A. Tarasevich (OSO 42-28-59-85 P), which corresponds to 5 × 10 ⁹ m.k. / ml for tularemia microbe. Then, it was diluted in a 0.9% sterile solution of sodium chloride so that the concentration of tularemia pathogens ranged from 1 × 10 ⁶ to 1 × 10 ¹ MK / ml. DNA was isolated from pure cultures of tularemia and heterologous microorganisms by nucleosorption and lysis of cells with guanidine thiocyanate using the DNA sorb-B kit (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor) in accordance with the instructions for use.

Given that the causative agent of tularemia belongs to agents of the pathogenicity group I, sample preparation and disinfection of the material for PCR was 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” and MU 3.1.2007-05 “Epidemiological surveillance of tularemia”.

The specificity of the designed primers was evaluated on a collection of 18 strains, of which 6 Francisella tularensis strains and 12 heterologous microorganism strains (4 strains of L. pneumophila and 1 strain of Yersinia pestis, Y. pseudotuberculosis, Bacillus anthracis, V. cholerae, Escherichia coli, B. suis, B. abortus, B. melitensis). Assessment of specificity showed the absence of amplification products with DNA of heterologous strains. FIG. 2 shows the result of an assessment of the specificity of oligonucleotide primers.

The sensitivity of the primers was evaluated using DNA samples isolated from ten-fold dilutions of the strain F. tularensis subsp. holarctica 15 NIIEG. An analysis of the results showed that the sensitivity of the designed primers was 1 × 10 ³ m.k. in the sample. Fig 3 and Fig 4. display the result of determining the sensitivity of oligonucleotide primers.

Thus, the developed primers can be used to detect the DNA of the tularemia causative agent and make it possible to quickly detect the tularemia causative agent in samples of pure cultures with high sensitivity and specificity. As a result of the assessment, it was found that the selected primers for the detection of tularemia pathogens by the loop isothermal reaction have 100% specificity and sensitivity - 1 × 10 ³ m.k. in the sample.

A set of oligonucleotide primers Ft 101 and a method for determining bacteria Francisella tularensis (Options)

Claims (3)

1. A set of oligonucleotide primers Ft 101 for determination of bacteria of the species Francisella tularensis by loop isothermal amplification, containing oligonucleotides SEQ ID No. 2; SEQ ID No. 3, SEQ ID No. 4; SEQ ID NO: 5, complementary to the ACP target gene of bacteria of the species Francisella tularensis encoded by the nucleotide sequence of SEQ ID NO: 1. 2. A method for determining bacteria of Francisella tularensis by the method of loop isothermal amplification (LAMP), characterized in that in the amplification reaction of a portion of the target ACPA target gene of bacteria of the species Francisella tularensis, a set of oligonucleotide primers according to claim 1 is used, wherein the synthesis and accumulation of fragments in one tube the target gene occurs at 60 ° C for a maximum of 60 minutes, and the determination of the target gene is carried out using agarose gel electrophoresis using a characteristic electrophoregram. 3. A method for determining bacteria of Francisella tularensis by loop isothermal amplification (LAMP) method, characterized in that in the amplification reaction of a portion of the target ACPA target gene of bacteria of the species Francisella tularensis, a set of oligonucleotide primers according to claim 1 is used, the synthesis and accumulation of fragments in one tube the target gene occurs at 60 ° C for a maximum of 60 minutes, and the target gene is determined by staining the amplification reaction products with the SYBRGreenI intercalating dye, and the amplification result each strain of F. tularensis is considered positive in case of bright green staining.

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