Method of detecting provirus of cattle leukosis

FIELD: medicine.

SUBSTANCE: claimed invention relates to the field of biotechnology and deals with a method of detecting a provirus of cattle leukosis. The characterised method includes the detection of LTR (Long Terminal Repeat) fragment - a sequence of the leukosis provirus. Determination of the size of an amplified fragment of a nucleotide sequence by the electrophoretic separation in an agarous gel is performed. As primers applied are oligonucleotides: BL1.F 5-GAGTTAGCGGCACCAGAAGC-3 and BL1.R 5-ATAGAGCTCGCGGTGGTCTC-3. The product of synthesis constitutes 175 pairs of the nucleotides. A genomic DNA is extracted by a sorbent method with the following elution in TE buffer, amplification is carried out in the mode: 95°C for 3 minutes 1 time, 94°C for 20 seconds - denaturation, 66°C for 20 seconds - annealing of the primers, 74°C for 25 seconds - elongation, 45 times, 74°C for 3 minutes - chains completion. As a positive control of reaction proceeding applied is a preparation of a positive control of BLV antigen.

EFFECT: invention can be applied in the molecular-genetic diagnostics of animal diseases and scientific research in veterinary.

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METHOD of DETECTING DNA PROVIRUS LEUKEMIA

The present invention relates to the field of biotechnology, molecular genetic diagnosis of animal diseases, research in veterinary science, molecular biology.

The bovine leukemia is a chronic infectious disease that is common worldwide. Called disease virus BLV (Bovine Leukemia Virus) and belongs to the genus Deltaretrovirus. By nature, it is similar to the virus HTLV and affects the bulk of B-lymphocytes. The virus leukemia cattle cause significant economic damage to the livestock farms of the Russian Federation.

In the complex of measures on improvement of herds of cattle from leukemia, the leading position belongs to the diagnosis. Issue timely and effective diagnosis of BLV in connection with extreme unfavorable epizootic situation on this disease and the lack of means of specific prophylaxis remains a critical issue.

One of the first and the main forms of diagnosis are the traditional methods of serological diagnosis of BLV (RIA, ELISA), which is not always possible to identify infected animals, and in some cases are unacceptable, for example in the early diagnosis of calves from seropositive parents that require the use of them in combination with methods mol�about genetic, in particular PCR, with which is possible early detection of BLV in dysfunctional leukemia farms, as well as increasing the efficiency and reducing the period of the anti-leukemic activities.

Polymerase chain reaction (PCR) is an effective and highly sensitive method, which is based on a multiple increase (amplification) of the studied fragment of the DNA sequence (target DNA) to the ability of its identification in gels or by using fluorescent dyes in real-time.

The specificity of the methods (NDP) is in the selection of oligonucleotide primers-primers, forward F and reverse R primers that define the scope elongation thermophilic polymerase. It is also important the selection amplificative fragment of proviral DNA of bovine leukemia.

The known method (Patent - US 6646116 B1, 11.11.2003, C07/H 21/04) differentiation gene variant TAX virus Laosa cattle, consisting in carrying out a panorama using primers capable of identifying the full sequence of the gene for further detection of mutations encoded by this gene amino acid sequences and identifying options for the TAX gene of BLV. In particular we used the following primers:

In tax2:5 AG TST AGA GCT GTC GAC TST GTC TG 3

In tax3:5 ACC TCG AGA TGG CAA GTG TTG TTG GTT GG 3.

Also known a method of detecting DNA provirus leukemia� cattle method pnrm. in the mode of real time (RT-PCR) and nested pnrm (nested PCR) (Log - Journal of Veterinary Diagnostic Investigation (J Vet Diagn Invest). 2003, Vol.15, 72-76. USA).

The proposed primers complementary to the POL gene have the following structure:

To use the panorama in real time together with primers to add to the reaction mixture was additionally synthesized POL probe (molecular beacon) 5 - (FAM)-

fluorescent label, which is the detection signal.

The method of nested PCR (nested PCR) involves the use of two pairs of primers - outer (1 round) and nested (2nd round). When both methods PCR product size is 202 nucleotide pairs. However, the known methods of detecting DNA of BLV provirus: in the analogue did not provide a detection area conservative Pol gene provirus.

A method of detecting DNA of bovine leukemia provirus Pol gene using the primers: RA 2: 5 - TGA ACG GAC AAA TGG ACT GCT C-3, P r2 5 - CCG ACA GAG AGC GAG GAG AG -3, giving the output product size 438 nucleotide pairs with subsequent visualization in 1.5% agarose gel in the presence of ethidium bromide as a dye (patent-2445370).

The drawback of all these methods is the requirement of a minimum number of copy number of sequences in the analyzed volume equal to 10 copies. The disadvantage is also the fact that while on study� persistent lymphocytosis titer of virus in the blood falls and may reduce the number of provirus copies below the claimed analytical sensitivity, which leads to false-negative results.

The object of the invention is to develop an effective method of detecting DNA provirus of bovine leukemia by PCR, allows for the increase of sensitivity for a selected region of DNA to detect the presence of the provirus in the diagnosis of Ural type BLV, with the ability to detect DNA provirus of bovine leukemia in the presence of a minimum number of copies of sequences in the analyzed volume (less than 10 copies), as well as enabling analysis at the stage of persistent lymphocytosis, at a lower titer of virus in the blood.

The task is solved in that using a straight F and reverse R primers for the detection of a fragment of the LTR (Long Termal Repeat) sequences of provirus bovine leucosis, amplification is carried out (building up) of the desired fragment, and determining the size amplificatoare fragment of the nucleotide sequence is carried out electrophoretic separation in an agarose gel, the primers are the oligonucleotides of the following structure: BL1.F 5-GAG TTA GCG GCA CCA GAA GC-3; BL1.R 5-ATA GAG CTC GCG GTG GTC TC 3, and the product of the synthesis of primers is 175 pairs of nucleotide and genomic DNA isolated sorbenty method, followed by elution in buffer TE (simple rapid method able� " s DNA), and amplification is carried out in solution in 80 mm Tris-HCL (pH 8.0), 0.1% Triton X-100, 24 mm (NH4)2SO4, 0.5 mm EDTA, 0.2 mm of each of the triphosphates, 0.5 mm of each primer and 1 unit of TAG polymerase and 40 ng of the analyzed genomic DNA of an infected animal, when the final volume of 20 µl solution, wherein the amplification is carried out in the regime: 95°C 3 minutes 1 time (process denaturation), 94°C - 20 seconds denaturation, 66°C - 20 seconds - annealing of primers, 74°C - 25 seconds - elongation, 45 times, completing circuits 74°C - 3 minutes, moreover, the detection of amplified fragments is carried out in 2% agarose gel in buffer DIDN, 0.5 mm with ethidium bromide, and as a positive control of PCR using the drug positive control antigen BLV.

Positive analysis of the confirm test systems domestic manufacturers.

In the process of the carried out works as a DNA target selected regions of the LTR (Long Terminal Repeat). The LTR region is found in all retroviruses and is required for integration of the provirus into the host genome region fenceroy provirus genome, thus increasing chopinot DNA target 5 two times, respectively, increases the sensitivity of the method that shows the obvious effect of the proposed method.

Then using the computer program Clustal W2 multi sequence alignment based on inform�tion is represented in the database of Internet resources NCBI GenBank were selected and analyzed 50, in our opinion, the most discordant genotype BLV to determine conservative areas of the virus. The selection of primers was performed using the program Lasergene V7.1 (USA). When conducting a computer analysis to the selected parcel LTR define basic criteria: a) the degree of homology of primers; b) lack semicomplete sites; b) the proximity of the melting temperature of the primers. We were picked up the following primers:

for the detection of LTR sequences of bovine leukemia provirus with the release of the product length 175 nucleotide pairs. Synthesis of oligonucleotide primers were ordered in the commercial center.

Fig. 1. presents the èlektroforegramme of amplification products M - token increments of 50 nucleotide pairs (50-500) " - "- healthy animals, " BL "+" - positive leukemia virus animals, With "-" and " + " for negative and positive controls.

Fig. 2. presents the èlektroforegramme of amplification products; M - marker increments of 50 BP (50-500), " BL "+" infected animals detected by the developed method, BLV "+" infected animals diagnosed using a diagnostic system of the Russian manufacturer, BIV test the specificity of the primers in the presence of virus DNA immunodeficita cattle, With "-" and "+" negative and positive controls.

For �technical confirmation of the possibility of early detection of bovine leukemia provirus in blood of infected animals were selected for the group.

A. Animals with an acute form of leukemia identified hematological method 28 goals.

Selected as the positive group.

B. a Group of youngsters at the age of 2-3 months after birth and is diagnosed on the subject of BLV infection by the method of REID (reaction immunodiffusion) showing a positive result.

Animals were selected on farms farming "Isimgready" Ishim district of the Tyumen region.

Both groups of subjects were studied by the method of the DAG, the data presented in table 1.

Animals in group B the number 4, which showed a negative result by PCR diagnosis were isolated and separated from the rest of the herd. Subsequent test analysis after 6 months confirmed prior and was negative. The presence of REED + result in young was the result of the influence kolostralnogo immunity twice and confirmed the test result pnrm analysis is considered in this case as the most accurate and informative method for early diagnosis of bovine leukemia provirus in calves.

This method is accurate, sensitive, with which you can more accurately in the early stages to identify a fragment of proviral DNA of leukemia virus of cattle in the biological material, in the presence of minimalno� the number of copies of the sequence in the analyzed volume at the stage of persistent limpieza, at low titer of virus in the blood.

A method for the detection of provirus bovine leucosis, including the identification of the fragment of the LTR (Long Terminal Repeat) sequences of leukemia provirus, determining the size of amplificatoare fragment of the nucleotide sequence by electrophoretic separation in agarose gel, the primers are the oligonucleotides of the following structure: BL1.F 5-GAGTTAGCGGCACCAGAAGC-3; BL1.R 5-ATAGAGCTCGCGGTGGTCTC-3, and the product of synthesis is 175 pairs of nucleotides, wherein the genomic DNA is isolated sorbenty method, followed by elution in buffer TE, and amplification is carried out in a solution of 80 mm Tris-HCL (pH 8.0), 0.1% Triton X-100, 24 mm (NH4)2SO4, 0.5 mm EDTA, 0.2 mm of each of the triphosphates, 0.5 mm of each primer, 1 unit of TAG polymerase and 40 ng of the analyzed genomic DNA of an infected animal, at finite solution volume of 20 μl amplification is carried out in the regime: 95°C 3 minutes 1 times, 94°C - 20 seconds denaturation, 66°C - 20 seconds - annealing of primers, 74°25 seconds elongation, 45, 74°C - 3 minutes - building up of the circuits, and detection of amplified fragments is carried out in 2% agarose gel DIDN buffer, 0.5 mm with ethidium bromide, and as a positive control of PCR using the drug positive control antigen BLV.



 

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