RU2706570C1 - SET OF OLIGONUCLEOTIDE PRIMERS Ft 40 AND A METHOD OF DETERMINING BACTERIA FRANCISELLA TULARENSIS (VERSIONS) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, molecular biology and medical microbiology. What is described is a set of oligonucleotide primers for the loop isothermal amplification reaction (LAMP), which enables to amplify bacteria-specific Francisella tularensis DNA fragments from the target gene having the nucleotide sequence SEQ ID No.1. Described set of oligonucleotide primers includes: FIP primer – "Ft40", having nucleotide sequence SEQ ID No. 2, BIP primer – "Ft40", having nucleotide sequence SEQ ID No. 3, primer F3 – "Ft40", having nucleotide sequence SEQ ID No. 4, primer B3 – "Ft40" having nucleotide sequence SEQ ID No. 5.

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

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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. To detect and identify 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 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 (genes chilf, RD1, 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, 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 Ft40-F3 F. tularensis);

SEQ ID No: 3 (reverse external primer Ft40-B3 F. tularensis);

SEQ ID No: 4 (direct internal primer Ft40-FIP F, tularensis);

SEQ ID No: 5 (reverse inner primer Ft40-BIP F. tularensis) for the method of 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 - 2 min, amplification 60 ° C - 60 min, the detection of the amplification product is carried out using agarose gel electrophoresis using a characteristic electrophoregram (option 1) or staining the products of the amplification reaction with intercalating dye SYBR Green I (option 2)

The technical result is also achieved by using the ISFtu2 gene as the target target, which encodes the insertion element, which is present in several copies only in F. tularensis.

Justification of the choice of oligonucleotide primers and target DNA.

The sequences of the insertion element ISFtu2, which is present only in this microorganism, were found in the genome of the tularemia microbe (Thomas R., Johansson A., Neeson B., Isherwood D., Sjostedt A., Ellis J., Titball RW. Discrimination of human pathogenic subspecies of Francisella tularensis by using restriction fragment length polymorphism. J. Clin. Microbiol. 2003.41: 50-57). The selected ISFtu2 gene is present in the genome of virulent for humans subspecies F. tularensis in several copies. The sequence of SEQ ID No. 1 (target gene ISFtu2) for the selection of primers for the detection of DNA of the causative agent of tularemia by loop isothermal amplification was obtained from GenBank NCBI database (National Center for Biotechnology Information, USA).

In the experiments, strains of F. tularensis were used, using disinfected suspensions of the microorganism in concentrations from 1 × 10 ⁶ m.k. / ml to 1 × 10 ¹ m.k. / ml for DNA extraction. The primers were tested on a set of strains of tularemia pathogens from the microorganism collection of the Irkutsk Research Anti-Plague Institute of Siberia and the Far East of Rospotrebnadzor.

The sensitivity of the amplification reaction with primers was evaluated by studying DNA samples isolated from ten-fold dilutions of pure cultures of tularemia pathogens.

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

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

Table 2. The results of amplification of DNA strains of the causative agent of tularemia with a set of LAMP primers SEQ ID No. 2; SEQ ID No. 3, SEQ ID No. 4; SEQ ID No. 5 using SD polymerase.

Table 3. Assessment of the specificity of the set of LAMP primers SEQ ID No. 2; SEQ ID No. 3, SEQ ID No. 4; SEQ ID No. 5.

Table 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. Used DNA from strains of F.tularensis ssp. holarctica I-393.

FIG. 1. The sequence of SEQ ID No. 1 is the nucleotide sequence of ISFtu2 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 and DNA heterologous strains.

Designations: 1 - DNA molecular weight marker (100-1000 bp), 2 - F. tularensis I-393 DNA, 3 - Y. pestis I-1251 DNA, 4 - Y. pseudotuberculosis I-226 DNA, 5 - DNA of Y. enterocolitica I-132, 6 - DNA of Escherichia coli 52, 7 - DNA of Bacillus anthracis I-362, 8 - negative control LAMP (water).

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.

Designations: 1-6: DNA was isolated using the Riboprep kit, 1 - DNA from a suspension of bacteria 10 ⁶ mk / ml, 2 - 10 ⁵ mk / ml, 3 - 10 ⁴ mk / ml, 4 - 10 ³ m.k. / ml, 5 - 10 ² m.k. / ml, 6 - 10 m.k. / ml.

8-12: DNA was isolated by thermolysis: 8 - DNA from a suspension of bacteria 10 ⁶ m.k. / ml, 9 - 10 ⁵ m.k. / ml, 10 - 10 ⁴ m.k. / ml, 11 - 10 ³ m .k./ ml, 12 - 10 ² m.k. / ml, 13 - 10 m.k. / ml. 14 - negative control LAMP (water), 7 - marker of molecular weight of DNA (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 of reaction products SYBR Green I.

Designations: 1-7: DNA was isolated by thermolysis: 1 - DNA from a suspension of bacteria 10 ⁶ m.k. / ml, 2 - 10 ⁵ m.k. / ml, 3 - 10 ⁴ m.k. / ml, 4 - 10 ³ MK./ml, 5 - 10 ² MK./ml, 6 - 10 MK./ml, 7 - negative control LAMP (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 744 bp region of the F.tularensis genome was selected to construct the direct and reverse external primers F3 and B3, the direct and reverse internal primers FIP and BIP. (GenBank NCBI, GeneID: 3192483) encoding an insertion element. To the plot of the gene SEQ ID No. 1 (Fig. 1) primers were 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. 2; SEQ ID No. 3, amounted to 201 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 online primer calculation program (http://primerexplorer.jp/lampv5e/index.html) was used. When selecting primers were guided by the requirements for the oligonucleotides used in LAMP. Analysis of the formation of secondary structures (dimers, hairpins) with the selected primers was performed using the mfold computer program (http://unafold.rna.albanv.edu/?q=mfold/DNA-Folding-Form). The 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 1x SD buffer (RusEnzyme, Russia), 40 units. SD polymerase (RusEnzyme, Russia), 3.5 mM MgCl ₂ , 0.5 mM each dNTP, 4 primers: 0.2 μM SEQ ID No. 2 (Ft40F3), 0.2 μM SEQ ID No. 3 (Ft40B3), 0.8 μM SEQ ID No. 4 (Ft40BIP), 0.8 μM SEQ ID No. 5 (Ft40FIP), 5 μl of the 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). Electrophoresis of LAMP products was performed on a 1.2% agarose gel in TAE buffer. Visualization was carried out on a UV transilluminator at a wavelength of 312 nm. (Option 1) The result of amplification 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). Table 2 displays the results obtained by amplification of DNA of strains of the causative agent of tularemia with a set of primers selected for the sequence of SEQ ID No. 1.

According to option 2, the LAMP products were detected by staining the amplifications with the SYBR Green I intercalating dye. The samples were stained immediately after the reaction by adding dye to the reaction to a final concentration of 10x. 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 bright green staining after adding S YBR Green I to the amplifications.

Example 3. Determination of the specificity and sensitivity 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 specificity and 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) ° C. 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. Two methods of DNA extraction were tested: thermoextraction (disinfected samples were heated at a temperature of 99 ° C in a solid-state thermostat "Termite" for 30 min) and DNA isolation using a set of reagents "Riboprep" (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 developed primers was evaluated on a collection of 15 strains, of which 10 strains of Francisella tularensis (Table 2) and 5 strains of heterologous microorganisms (Yersinia pestis, Y. pseudotuberculosis, Y. enterocolitica, Escherichia coli, Bacillus anthracis) (table 3). Assessment of specificity showed the absence of amplification products with DNA of heterologous strains (Fig. 2).

The sensitivity of the primers was evaluated using DNA samples isolated from ten-fold dilutions of F. tularensis strains. Analysis of the results showed that the sensitivity of the designed primers was 1 × 10 ³ mk / ml (table. 4, Fig. 3, Fig. 4).

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./ ml.

Claims (3)

1. A set of oligonucleotide primers Ft40 for the determination of bacteria 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 target lSFtu2 target gene of bacteria of the species Francisella tularensis, encoded by the sequence 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 gene target of the ISFtu2 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 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 ISFtu2 target gene of bacteria of the species Francisella tularensis, the oligonucleotide primer set 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 target target gene is determined by staining the amplification reaction products with the SYBRGreenI intercalating dye, and the amplification result is each strain of F. tularensis is considered positive in case of bright green staining.

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