RU2289627C2 - METHOD AND KIT FOR DETECTION OF LYME BORRELIOSIS BACTERIA (Borrelia burgdorferi) DNA - Google Patents

Abstract

FIELD: medicine diagnosis, molecular biology.

SUBSTANCE: invention relates to method for detection of DNA of human Lyme borreliosis excitant in peripheral blood, cell culture and mites. Claimed method includes DNA isolation from biological material; synthesis of primers; PCR with using synthesized primers and evaluation of obtained data. In method BBs14.F/BBs14.R - 5'-AAGAATACATTAAGTGCGATANN-3', 5'-CAATCCACTTAATTTTGTGTTAT-3' primers to gene site encoding 16S rRNA are used. Kit for this method also in disclosed.

EFFECT: high-specific, sensitive and simplified method.

2 cl, 2 dwg, 5 tbl

Description

The invention relates to molecular biology, genetic engineering, medicine: hematology, medical diagnostics and epidemiology, specifically to a method and kit for detecting bacterial DNA of Lyme borreliosis infectious diseases in peripheral blood, cell culture and ticks, and can be used in research institutions, regional, regional sanitary and epidemiological surveillance services and enterprises of the biological industry.

Recently, the polymerase chain reaction (PCR) method has been widely used to diagnose human viral and bacterial diseases and to indicate their pathogens, in particular, the indication of Borrelia burgdorferi (1).

The closest method and kit for detecting bacterial DNA is the method and kit for detecting bacterial DNA of Lyme borreliosis disease - Borrelia burgdorferi by PCR (2). The method is carried out as follows: a DNA fragment of size 840 nucleotides (bp) isolated from the conservative region of the Borrelia burgdorferi genome is partially sequenced and six primers are selected based on this sequence, which allow amplification of specific DNA fragments. To identify these products by the method of molecular hybridization, an additionally synthesized oligonucleotide, as well as a recombinant plasmid containing this fragment, were used as DNA probes.

The kit contains a positive control (recombinant plasmid), a negative control, 6 primers, a solvent and PCR oil, thermostable Tag polymerase enzyme, a reaction buffer, and a mixture of four deoxynucleotide triphosphates.

The disadvantages of this method and kit is that the PCR with six primers is difficult and increases the cost of analysis, does not make it possible to detect all genotypes related to the Lyme borreliosis bacterium - Borrelia burgdorferi, is used only for experimental studies to detect the DNA of the Lyme borreliosis Borrelia burgdorferi bacterium and only in cell culture.

The objective of the invention is the creation of a highly specific, sensitive, but simpler and faster method and an appropriate kit for detecting the DNA of the Lyme borreliosis bacterium Borrelia burgdorferi in human peripheral blood samples, cell culture and ticks using two primers, which allows visual assessment of pathogens in the analyzed material and detect all genes related to Borrelia burgdorferi sensu lato.

The task is achieved by a method for detecting the DNA of the Lyme borreliosis bacterium - Borrelia burgdorferi in human peripheral blood samples, cell culture and ticks, in which two primers BBsl4.F / BBsl4.R - 5'-AAGAATACATTAAGTGGGATATT-3 ′ were selected and synthesized for PCR 5'-CAATCCACTTAATTTTTGTGTTAT-3 ', encoding 16S rRNA, which is a component of the ribosome, with an annealing temperature of 51 to 52 degrees C for 10-30 seconds with the number of amplification cycles from 35-42 and a set including plastic tubes containing: primers BBsl4.F / BBsl4.R-5'-AAGAATACATTAAGTGCGATATT-3 ', 5'-CAATCCACTTAATTTTT GTGTTAT-3 'encoding 16S rRNA, which is a component of the ribosome; thermostable enzyme Tag polymerase; 5x reaction buffer; a mixture of four deoxynucleotide triphosphates; positive control - recombinant plasmid, negative control and PCR oil.

The invention.

A method for detecting the DNA of the pathogen Borrelia burgdorferi is as follows

Isolation of DNA from biological material

Isolation is carried out according to a standard method using proteinase K, extraction with phenol-chloroform and subsequent DNA precipitation with ethanol (3)

Samples (human peripheral blood samples, cell culture and ticks) are homogenized in 5-6 volumes of buffer A (10 mM Tris-HCl, pH 7.5; 10 mM NaCl, 3 mM MgCl2) and centrifuged at 2500 g for 10 min at 4 ° C. The precipitate was washed 2 times with buffer A, then resuspended in buffer B (10 mm EDTA, 100 ml NaCl, 50 mm Tris-HCl pH 8.5. 0.5% SDS, 200 μg / ml proteinase K) and incubated for 2 hours at a temperature 37 ° C. After standard extraction with phenol and chloroform, the DNA is precipitated with ethanol and dissolved in water to a concentration of 0.5 μg / ml.

Primer Synthesis

The synthesis of oligonucleotides is carried out by the traditional phosphoamitide method (4). For example, on a DNA synthesizer ASM-800 company "BIOSET".

PCR

PCR is carried out according to the standard method using two primers: BBsl4.F / BBsl4.R - 5'-AAGAATACATTAAGTGCGATATT-3 ', 5'-CAATCCACTTAATTTTTGTGTTAT-3', encoding 16S rRNA, which is a component of the ribosome selected according to the method described below. The primers are annealed at a temperature of 51-52 ° C for 10-30 seconds with the number of amplification cycles equal to 35-42.

DNA detection

Detection is carried out in a 2% agarose gel using TBE buffer (5.4 g Tris, 2.75 g boric acid, 0.46 g EDTA, dH ₂ O up to 1 L, pH 8.3) with ethidium bromide (2 μl / 100 ml of buffer). 10 μl of amplification (5) are added to the gel well under the buffer. Electrophoresis is carried out at a voltage of 20 V / cm for 10 minutes The results are visualized on a video system, for example, BIOTEST-D (Moscow).

Justification of the advantages of choosing primers

The rrs gene encoding 16S rRNA, which is a component of the ribosome, was selected as a unique sequence for the diagnosis of B.burgdorferi sensu lato. The following DNA sequences were analyzed (Table 1) obtained in the Genome Net - DNA Database of the National Institute of Genetics (Kyoto, Japan).

Primers were selected using the Primer Premier - V.5 program (Premier Biosoft International-1). For the plot U03396 (rrs gene, GenBank), several pairs of primers were selected (Table 2). Specific primers for B.burgdorferi were obtained as a result of the design of the 16S rRNA sequence and the 16S rRNA – 23S rRNA spacer region.

The specificity of the primers for 16S rRNA was evaluated by multiple sequence comparison using software such as BLAST for optimal assessment of species specificity and Clastal W for assessing polymorphisms in the compared sequences.

The optimal pair of primers (taking into account their thermodynamic characteristics) were chosen primers BBsl1.F / BBsl1.R and BBsl4.F / BBsl4.R (Table 3).

Primers BBsl1.F / BBsl1.R are located on the site of the rrs gene encoding 16S rRNA and initiates amplification of the product 537 bp Primers BBsl4.F / BBsl4.R are located on the spacer region 16S-23S and initiate amplification of the product 688 bp (figure 1).

These primers do not form secondary structures (hairpins) and dimers, which increases the efficiency of their binding to matrix DNA. However, the primer BBsl1.F has false priming sites, but without product formation.

Primer BBsl1.R. also has a false priming section to form products. However, the energy ΔG, which is formed during the complementary interaction, is below critical (ΔGk = -9). Primers BBsl4.F / BBsl4.R can form unstable cross-dimers.

Justification of the selection of conditions for PCR

The optimization of the amplification temperature was carried out experimentally, while maintaining all the conditions for the PCR formulation, the annealing temperature of the primers was changed from 45 ° C to 57 ° C.

At annealing temperature of primers of 45 ° С, DNA of all studied Borrelia strains was amplified; DNA of microorganisms of other taxonomic groups was not amplified under these conditions. When the temperature rises above 56 ° C, PCR products in the gel are not detected. The optimal ratio of the number of specific and non-specific products of the amplification of the DNA of the bacterium B.burgdorferi was established at annealing temperature of primers 52 ° C for a pair of primers BBsl1.F / BBsl1.R and 51 ° C for BBsl4.F / BBsl4.R!

When using the active temperature control mode, the following temperature range was revealed: the denaturation and elongation time was from 10 to 30 seconds, the optimal time was 15 seconds, the primer annealing time was from 10 to 30 seconds, the effective number of cycles was 35-42.

Analytical Characteristics

The analytical sensitivity of PCR was determined by the method of limiting dilutions of DNA of the bacterium Borrelia burgdorferi isolated from the culture. As a result of the assessment, it was found that the primers used in this study BBsl4.F / BBsl4.R - 5'-AAGAATACATTAAGTGCGATATT-3 ', 5'-CAATCCACTTAATTTTTTGTGTTAT-3', encoding 16S rRNA, which is a component of the ribosome, have high analytical sensitivity the level of 20 genomic equivalents in each PCR reaction (figure 2; Table 4).

A preliminary assessment of the analytical specificity of the primers used using the BLAST on-line server (blastn) showed the lack of homology of the selected sequence with other types of microorganisms, with a significance level of E = 10. (figure 2).

As a result, amplification with primers BBsl4.F / BBsl4.R (spacer region 16S-23S rRNA) in all cases yielded negative results. While the use of the BBsl1.F / BBsl1.R pair (16S rRNA) led in 40% of cases to weakly positive results with Preponema palidum, which served as the basis not to use this pair of primers in further testing.

Lyme borreliosis disease bacterial DNA detection kit - Borrelia burgdorferi includes plastic tubes containing thermostable Tag polymerase enzyme, a 5-fold reaction buffer, a mixture of four deoxynucleotide triphosphates, a positive control - a recombinant plasmid containing the bacterial flagelin gene fragment, oil for PCR and primers BBsl4.F / BBsl4.R - 5'-AAGAATACATTAAGTGCGATATT-3 ', 5'-CAATCCACTTAATTTTTTGTGTTAT-3', encoding 16S rRNA, which is a component of the ribosome.

The offered kit is equipped with 5 plastic test tubes.

The kit is designed to detect the DNA of Borrelia burgdorferi bacteria in cell culture, blood serum or tick by polymerase chain reaction with visual indication of the obtained amplicon DNA by gel electrophoresis in agarose and can be used in clinical and epidemiological studies. The kit is designed for 110 tests, including positive and negative controls.

The set works as follows.

PCR

1. Prepare tubes for amplification by numbering them and positioning them accordingly in a tripod. Two test tubes are prepared for controls: one for the negative and one for the positive control.

2. Prepare a mixture of components for amplification per sample: thermostable Tag polymerase enzyme, 5-fold reaction buffer, a mixture of four deoxynucleotide triphosphates and primers BBsl4.F / BBsl4.R - 5'-AAGAATACATTAAGTGCGATATT-3 ', 5' -CAATCCACTTAATTTTTGTGTTAT-3 'encoding 16S rRNA, which is a component of the ribosome.

3. The mixture was pipetted and 15 μl was added to each tube for amplification.

4. Pipette one drop of PCR oil into each tube using a pipette with a tip of up to 1 ml.

5. Pipette 10 μl of the DNA extracted from the clinical sample into the corresponding tube for amplification using an individual filter tip. Pipette 10 μl of control DNA into the tube labeled as positive control and 10 μl of the negative control into the tube labeled negative control.

6. The tubes are closed and centrifuged for 5 seconds at 3 thousand rpm

7. Transfer the tubes to a programmable thermostat (thermocycler), warm them up at 95 ° С for 5 min, and carry out amplification according to the following program (Table 5).

Analysis of diagnostic results

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

2. In a positive control sample for Borrelia burgdorferi, a strip of 500 nucleotide pairs is detected.

3. In the negative control sample, the band corresponding to the genome fragments of these infections should be absent. The appearance of a band in the negative control indicates contamination of the kit.

In the analyzed samples, the absence of a band strictly at the level of the corresponding control indicates the absence of a pathogen in the sample; the presence of a band corresponding to a positive control in electrophoretic mobility indicates the presence of this pathogen in the sample. The results can be documented by photographing gels using an orange or interference filter.

Thus, the proposed method and kit have novelty: species-specific sites of the genome of the bacterium of the Lyme borreliosis disease - Borrelia burgdorferi in the region of the gene encoding 16S rRNA (rrs) have been established; optimal annealing temperature: denaturation and elongation time was from 10 to 30 sec, optimal 15 sec, annealing time for a pair of 16S rRNA primers from 10 to 30 sec at annealing temperature of 51-52 ° С, the number of amplification cycles 35-42, which allows to achieve high sensitivity using two primers instead of six used in standard PCR, reduce the time for bacterial DNA detection, detect up to 10 femtograms of Borrelia burgdorferi bacteria DNA or 20 particles in 1 ml of infectious material, including blood samples and ticks, within 2 hours.

Information sources

1. Yigong Ge, J Bacteriol, Mau 1998, p. 2418-2425, Vol. 180, No.9.

2. Dionysios Liveris, Journal of Clinical Microbiology, March 1999, p. 565-569, Vol. 37, No.3.

3. Manniatis T., 1984.

4. Grossman L., 1987.

5. Osterman L.A., 1981.

Table 1 The name of the microorganism Registration number (Genbank) Gene Strain opening date Gene Size (Mon) Gb_ba: Bbrnaopr U03396 Rrs (16S rRNA) B31 10/93 11955 Gb_ba: Vbu44938 U44938 Rrs (16S rRNA) 5MT 5/96 1,533 Gb_ba: Bor16rg L39080 rrs (16S rRNA) 9MT 3/95 1,533 Gb_ba: Vbu44939 U44939 rrs (16S rRNA) 917Y 5/96 1,533 Gb_ba: Bb16s297 X85204 rrs (16S rRNA) 297 5/95 1,488 Gb_ba: Borrrd L36160 rrs (16S rRNA) 934U 9/94 1,536 Gb_ba: War16rga L39081 rrs (16S rRNA) 935T 3/95 1,542 Gb_ba: Borrrdq M64309 rrs (16S rRNA) 1352 4/92 1,481 Gb_ba: Borrrd M64310 rrs (16S rRNA) 20004 4/92 1,480 Gb_ba: Bb16srrna X57404 rrs (16S rRNA) B31 3/92 1,465 Gb_ba: Borssrna M59293 rrs (16S rRNA) B31 4/92 1,480 Gb_ba: Borrnaca M89935 rrs (16S rRNA) CA2-87 1/93 1,291 Gb_ba: Bb16sdk7 X85195 rrs (16S rRNA) DK7 5/95 1,488 Gb_ba: Bb16sdk29 X85202 rrs (16S rRNA) DK29 5/95 1,488 Gb_ba: Bb16sdunk X85201 rrs (16S rRNA) DUNKIRK 5/95 1,488 Gb_ba: Bbu28501 U28501 rrs (16S rRNA) ESP-1 7/95 1,488 Gb_ba: Warrr16sa M60967 rrs (16S rRNA) G2 4/92 1,483 Gb_ba: Borrnail M89936 rrs (16S rRNA) Illinois 1 1/93 1,291 Gb_ba: Bb16skipp X85196 rrs (16S rRNA) Kipp 5/95 1,488 Gb_ba: Bb16slipz X85203 rrs (16S rRNA) Lipipz 5/95 1,488 Gb_ba: Warrr16sc M60969 rrs (16S rRNA) Sh-2-82 4/92 1,476 Gb_ba: Borrnavs M89938 rrs (16S rRNA) VS219 1/93 1,350 Gb_ba: Borrgda L40596 Rrs (16S rRNA) 3/95 1,492

table 2 Primer Locus Nucleotide sequence BBsl1.F 19-44 bp 5'-CAGACAACAGAAGGGAATTTAAATG-3 ' BBsl1.R 532-556 bp 5'-CAAGTGATGTATTAGCATCAACTG-3 ' BBsl2.F 87-111 bp 5'-TTGTTAGCAGGAGTATGCAATATC-3 ' BBsl2.R 408-429 bp 5'-GGGCTTGTAAGCTCTTTAACT-3 ' BBsl3.F 128-149 bp 5'-GCTGCTGGCATGGGGGTTTCT-3 ' BBsl3.R 729-754 bp 5'-CAATAGCATACTCAGTAGTACTATT-3 ' BBsl4.F 1224-1248 bp 5'-AAAGAATACATTAAGTGCGATATT-3 ' BBsl4.R 1888-1912 bp 5'-CAATCCACTTAATTTTTGTGTTAT-3 ' Table 3 Primer Gene Length Sequence Number Gc% Tm (° C) Ta Ppt (° C) BBsl1.F rrs 25 19 52.6 55.1 BBsl1.R rrs 24 532 60.0 61.3 Product 1 537 45.4 87.7 53.0 BBsl4.F rrs 24 1224 54.5 59.5 BBzl4.R rrs 24 1888 50,0 61.9 Product 2 688 34.5 83.3 51.1

Table 4 Test strain Primer B.Bsl1.F / B.Bsl1.R GE in the sample Primer B.Bsl4.F / B.Bsl4.R GE in the sample B.V. s.s. B31 120 150 B.afzelii 600 300 B.V.s.s. Taldom No. 17 600 300 Table 5 Matrix-controlled amplifiers: "MiniCicler" type Amplifiers with active regulation (in vitro): type "Tertsik", "Omn-E" Temperature Time Cycles Temperature Time Cycles one 95 ° 5 minutes one 95 ° 5 minutes one 2 95 ° 1 min 40 95 ° 30 s 40 52 ° 1 min 52 ° 30 s 72 ° 1 min 72 ° 30 s 3 72 ° 1 min one 72 ° 1 min one four 10 ° Store 10 ° Store

Claims (2)

1. A method for detecting the DNA of the Lyme borreliosis disease bacterium - Borrelia burgdorferi, including the isolation of DNA from biological material, the synthesis of primers, the formulation of the polymerase chain reaction using synthesized primers and the evaluation of the reaction by gel electrophoresis in 2% agarose, characterized in that The polymerase chain reaction is used to detect bacterial DNA in cell culture, samples of human peripheral blood and ticks two primers: BBs14.F / BBs14.R 5'-AAGAATACATTAAGTGCGATATT-3 ', 5'-CAATCCACTTAATTTTTGTGTTAT-3 ', to the site of the gene encoding 16S rRNA, which is a component of the ribosome, primers are annealed at a temperature of 51-52 ° С for 10-30 s, and the number of amplification cycles is 35-42, while the absence of a band strictly at the level of the corresponding control indicates the absence of the pathogen in the sample, and the presence of a band corresponding to a positive control in electrophoretic mobility indicates the presence of this pathogen in the sample. 2. A kit for detecting the DNA of the Lyme borreliosis disease bacterium - Borrelia burgdorferi, which includes thermostable Tag polymerase enzyme, a reaction buffer, a mixture of four deoxynucleotide triphosphates, a positive control - a recombinant plasmid containing the bacterium flagelin gene fragment, and an oil for polymerase chain reaction in that it contains primers: BBs14.F / BBs14.R-5'-AAGAATACATTAAGTGCGATATT-3 ', 5'-CAATCCACTTAATTTTTGTCTTAT-3', encoding 16S pRNA, which is a component of the ribosome and a 5-fold buffer for the formulation of the reaction.

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