RU2558252C2 - Method of detecting provirus of cattle leukosis - Google Patents

Abstract

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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Description

METHOD FOR DETECTING LEUKOSIS PROVIRUS DNA

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

Bovine leukemia is a chronic infectious disease common throughout the world. The disease is caused by the BLV virus (Bovine Leukemia Virus) and belongs to the genus Deltaretrovirus. By its nature, it is similar to the HTLV virus and mainly affects B-lymphocytes. The cattle leukemia virus causes significant economic damage to livestock farms of the Russian Federation.

In the complex of measures for improving cattle herds from leukemia, the leading place belongs to diagnostics. The problem of timely effective diagnosis of VLCCR due to the extremely unfavorable epizootic situation for this disease and the lack of specific prophylaxis is still acute.

One of the first and main forms of diagnosis is the traditional methods of serological diagnosis of VLCC (RID, ELISA), which do not always allow the detection of infected animals, and in some cases are unacceptable, for example, in the early diagnosis of young animals from seropositive parents, which requires their use in combination with molecular genetic methods, in particular PCR, with the help of which early detection of VLCRC in leukemic dysfunctional farms is possible, as well as increasing efficiency and reducing the time for rotivoleukemic events.

Polymerase chain reaction (PCR) is an effective and highly sensitive method, which is based on the multiple increase (amplification) of the studied fragment of DNA sequences (target DNA) until it can be identified in gels or using real-time fluorescent dyes.

The specificity of the methods (PNR) consists in the selection of oligonucleotide primer seeds, direct F and reverse R primers, which form the elongation region of thermophilic polymerase. The selection region of the amplified fragment of cattle leukemia proviral DNA is also important.

There is a method (Patent - US 6646116 B1, 11.11.2003, C07 / H 21/04) for differentiating a variant of the TAX gene of cattle leiosis virus, which consists in conducting a NDP using primers capable of detecting the complete sequence of a gene to further identify mutations encoded by this gene amino acid sequences and determination of variants of TAX gene for VLCC. In particular, the following primers were used:

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

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

There is also known a method for detecting bovine leukemia provirus DNA by real-time PNR (RT-PCR) and nested PCR (nested PCR) (Journal of Veterinary Diagnostic Investigation (J Vet Diagn Invest). 2003, Vol.15, 72 -76. USA).

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

To use the real-time PNR method, it is necessary, together with primers, to add to the reaction mixture an additionally synthesized POL probe (molecular beacon) 5 - (FAM) -

fluorescent label, due to which the signal is detected.

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

There is a method of detecting cattle leukemia provirus DNA by the Pol gene where primers are used: 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 followed by visualization in a 1.5% agarose gel in the presence of ethidium bromide as a dye (patent-2445370).

The disadvantage of all these methods is the need for a minimum number of copies of sequences in the analyzed volume equal to 10 copies. The disadvantage is the fact that being at the stage of persistent lymphocytosis, the titer of the virus in the blood drops and it is possible to decrease the number of copies of the provirus below the declared analytical sensitivity, which leads to false negative results.

The objective of the invention is to develop an effective method for detecting DNA of cattle leukemia provirus by PCR method, which allows detecting the presence of provirus by diagnosing the Ural type of VLCC, with the possibility of detecting cattle leukemia provirus DNA in the presence of a minimum number of sequence copies in the analyzed volume (less than 10 copies), as well as the possibility of analysis at the stage of persistent lymphocytosis, with transpose titer of virus in the blood.

The problem is solved in that using direct F and reverse R primers to identify the LTR fragment (Long Termal Repeat) - the sequence of the bovine leukemia provirus, the desired fragment is amplified (expanded), and the size of the amplified fragment of the nucleotide sequence is determined by electrophoretic separation in agarose gel while oligonucleotides of the following structure are used as primers: BL1.F 5-GAG TTA GCG GCA CCA GAA GC-3; BL1.R 5-ATA GAG STS GCG GTG GTC TC 3, and the product of the synthesis of primers is 175 pairs of nucleotides, and the genomic DNA is isolated by the sorbent method followed by elution in TE buffer (simple express method for DNA isolation), and amplification is carried out in solution: 80 mM Tris-HCL (pH 8.0), 0.1% Triton X-100, 24 mM (NH ₄ ) ₂ SO ₄ , 0.5 mM EDTA, 0.2 mM of each triphosphate, 0.5 mM of each primer and 1 unit of TAG polymerase and 40 ng of the analyzed genomic DNA of an infected animal, with a final solution volume of 20 μl, while amplification is carried out in the mode: 95 ° C - 3 minutes 1 time (process ), 94 ° С - 20 seconds - denaturation, 66 ° С - 20 seconds - annealing of primers, 74 ° С - 25 seconds - elongation, 45 times, extension of chains 74 ° С - 3 minutes, and amplified fragments are detected in 2 % agarose gel in TBE buffer, 0.5 mM ethidium bromide, and as a positive control for the passage of the polymerase chain reaction, a drug for the positive control of VLCC antigen is used.

A positive analysis is confirmed by the test systems of domestic manufacturers.

In the course of the work, the LTR (Long Terminal Repeat) region was selected as the target DNA. The LTR region is contained in all retroviruses and is necessary for the integration of the provirus into the host genome; the region flanks the provirus genome, thus increasing the copy number of the target DNA 5 twice, and the sensitivity of the method increases accordingly, which shows an unobvious effect of the proposed method.

Then, using the computer program Clustal W2 multi sequence alignment based on the information provided in the GenBank NCBI Internet resources database, we selected and analyzed the 50, in our opinion, most discordant VLCCR genotypes for the determination of conservative areas of the virus. Primers were selected using the Lasergene V7.1 software (USA). When conducting computer analysis, the main criteria are determined for the selected LTR site: a) the degree of homology of the primers; b) lack of self-complementary sites; c) the proximity of the melting temperature of the primers. We have selected the following primers:

for detecting the LTR sequence of cattle leukemia provirus with a product yield of 175 nucleotide pairs in length. The synthesis of oligonucleotide primers was ordered at a commercial center.

In FIG. 1. The electrophoregram of amplification products is presented. M - marker in increments of 50 pairs of nucleotides (50-500), K "-" - healthy animals, BL "+" - animals infected with leukemia virus, C "-" and C "+" - negative and positive controls.

In FIG. 2. The electrophoregram of amplification products is presented; M - marker in steps of 50 bp (50-500), BL "+" infected animals diagnosed by the developed method, BLV "+" infected animals diagnosed using the diagnostic system of the Russian manufacturer, BIV primer specificity test in the presence of cattle immunodeficiency virus DNA, C "-" and C " + "Negative and positive controls.

Groups were selected for technical confirmation of the possibility of early detection of cattle leukemia provirus in the blood of infected animals.

A. Animals with an acute form of leukemia revealed by the hematological method of 28 goals.

Selected as a positive group.

B. A group of young animals at the age of 2-3 months after birth and diagnosed for VLCRS infection by the RID method (immunodiffusion reaction), which showed a positive result.

Animals were selected on the farms of the Ishimagroproduct farm of the Ishim district of the Tyumen region.

Both groups were investigated by the test method GPAR, the data are presented in table 1.

Animals of group B in an amount of 4, which showed a negative result for PCR diagnostics, were isolated and kept separately from the rest of the herd. Subsequent test analysis after 6 months confirmed the previous one and was negative. The presence of the RID + result in young animals was the result of the influence of colostral immunity, and the twice-confirmed test result of the NDP analysis is considered in this case as the most accurate and informative method for the early diagnosis of cattle leukemia provirus in calves.

This method is reliable, highly sensitive, with the help of which it is possible to more accurately detect a fragment of proviral DNA of cattle leukemia virus in biological material in the early stages, with a minimum number of copies, the sequence in the analyzed volume at the stage of persistent lymphocytosis, with a reduced titer of the virus in the blood.

Claims (1)

A method for detecting bovine leukemia provirus, comprising detecting a LTR fragment (Long Terminal Repeat) - a leukemia provirus sequence, determining the size of an amplified fragment of a nucleotide sequence by electrophoretic separation in an agarose gel, using oligonucleotides of the following structure as primers: BL1.F 5- GAGTTAGCGGCACCAGAAGC-3; BL1.R 5-ATAGAGCTCGCGGTGGTCTC-3, and the synthesis product is 175 base pairs, the genomic DNA is isolated by the sorbent method, followed by elution in TE buffer, and amplification is carried out in a solution of 80 mM Tris-HCL (pH 8.0), 0.1% Triton X-100, 24 mM (NH ₄ ) ₂ SO ₄ , 0.5 mm EDTA, 0.2 mm each of triphosphates, 0.5 mm each primer, 1 unit of TAG polymerase and 40 ng of the analyzed genomic DNA of an infected animal, the final volume of the solution is 20 μl, amplification is carried out in the mode: 95 ° C - 3 minutes 1 time, 94 ° C - 20 seconds denaturation, 66 ° C - 20 seconds - annealing of primers, 74 ° C - 25 seconds prolongation, 45 times, 74 ° С - 3 minutes — chain completion, and amplified fragments were detected in 2% agarose gel in TBE buffer, 0.5 mM ethidium bromide, and the antigen positive control drug was used as a positive control for polymerase chain reaction VLKRS.

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