Diagnostic technique for cattle leukemia virus tolerance

FIELD: medicine.

SUBSTANCE: method involves allele-specific Nested-PCR with primers which are matched with nucleotide sequences coding amino acids in positions 70-71 of the amino acid sequence. The allele-specific primers E70f1 - 5'-AGAAGGAGATCCTGGAGGATAG - 3' and R71r1 - 5'-CCTGTCCACCTCGGCCCGCCTATC - 3' are matched with a part of BoLA-DRB3 gene located on chromosome 23 (localisation 23q21). They interact only with the nucleotide sequences coding alleles *11, *23, *28 =*7A causing genetic stability to cattle leukaemia. Then sequencing primer Zond 70/71 5'-GCCCGGCTACACCTGT - 3' is used to identify homo- or heterozygosity of an individual by the given alleles. If observing the primers interacting with alleles *11, *23, *28 =*7A, animals are considered to be leukaemia stable, while the absence of interaction with the same alleles can enable to refer to leukaemia unstable, and to neutral.

EFFECT: invention can be used for mass genetic typing of BoLA-DRB3 leukaemia tolerable animals in livestock and commodity economies for animal selection in a nuclear stock.

2 dwg, 4 tbl, 2 ex

 

The invention relates to biotechnology and molecular genetics, in particular to methods of gene diagnostics resistance of cattle to leukemia.

There is a method of determining the resistance of cattle to leukemia, which consists in the amplification of exon 2 of the gene BoLA-DRB3 (size PCR product is 284 BP) and subsequent analysis of restriction polymorphism of this region using serial processing endonuclease Rsa I, Hae III, Bst YI (Van Eijk, M.J., .Stewart, J.A.Haynes, and H.A.Lewin. 1992. Extensive polymorphism of the BoLA-DRB3 gene distinguished by PCR-RFLP. Anim. Genet. 23:483-496).

There is a method of determining the resistance of cattle to leukemia, which consists in the amplification of exon 2 of the gene BoLA-DRB3 and subsequent analysis of restriction polymorphism of this region by processing restrictase Rsa I, Hae III, BstYI, Bst2UI (kowaluk, NV and other Selection-genetic method of creating highly productive and resistant to the persistent limfotsitoz cattle. / Kowaluk NV, Shostak, VA, Satsuk V.F., Fomenko A.I // Bulletin of the RAAS. - 2005. No. 6. - P.67-69).

The main advantage of these methods is the ability to identify each specific allele of the previously described detection of new alleles.

The obvious drawback of this analysis is the complexity of the differentiation of genotypes, because the restriction fragments may differ from each other neznacit the flax. In addition, these methods require a lot of time and money on analysis.

There is a method of determining the resistance of cattle to leukemia, taken as a prototype, consisting of allele-specific PCR with primers selected for nucleotide sequences coding for amino acids Glu-Arg at positions 70-71 amino acid sequences and the amino acids Val-Asp-Thr-Tyr at positions 75-78 (J Immunol. 1993 Dec 15; 151(12):6977-85. Polymorphism in BoLA-DRB3 exon 2 correlates with resistance to persistent lymphocytosis caused by bovine leukemia virus. Xu A., van Eijk M.J., Park, C., Lewin H.A.).

This method is faster and cheaper than PCR-RFLP analysis, it also allows you to share all of the results into three groups: stable, sensitive and neutral alleles, rather than selecting each specific allele.

However, this method of analysis does not allow to identify Homo - or heterozygosity individuals on detected alleles.

When creating the present invention, the task was to develop a method of diagnosing the resistance of cattle to leukosis virus, providing a mass screening of the population in order to identify animals-carriers of alleles that provide resistance to leukemia.

The technical result of the invention is achieved by a method of diagnosing the resistance of cattle to leukosis virus, including the allele with ecifica Nested-PCR with primers matched to the positions 70-71 in amino acid sequence, wherein the primers are selected so that, identify animals-media only-resistant leukemia alleles, with subsequent determination of Homo - or heterozygosity of these animals according to the alleles by persecutione.

Total for the prototype and the present invention is the use of Nested-PCR with allele-specific primers.

Unlike the prototype of the invention involves mass screening of the population in order to identify using allele-specific PCR animals carrying only desirable alleles (i.e. alleles determining resistance to leukemia), and the subsequent persecutione found samples to establish a Homo - or heterozygosity of individuals on these alleles. The animals that carry sensitive or neutral alleles, not divided into 2 groups.

The prepared PCR products (amplicons) hybridized with sequanorum primer and diagnose the nucleotide sequence of the gene in real-time and simultaneous determination of 4 amino acids (E, R, Q, K) with the use of technology persecutione and identification of Homo - or heterozygosity for alleles determining resistance or susceptibility to the virus leukemia.

Example 1. Oligonucleotide primers were kindly the us on the basis of gene sequence BoLA-DRB3. They amplified fragments of the gene BoLA-DRB3 cattle in length 284 base pairs (BP), 233 BP and 93 BP (gene Bank M, M, M, M, M, M) in two stages. In the first stage, we used the primers l30 and l31 Protocol (table 1.1), in the second stage - l30, Bola32, E70fl and R71rT(the Protocol in table 1.3);

l30 - 5'-ATCCTCTCTCTGCAGCACATTTCC-3'

Bola31 - 5'-TTAAATTGCGCTCACCTCGCCGCT-3';

Bola32 - 5'-TCGCCGCTGCCACAGT-3';

E70f1 - 5'-AGAAGGAGATCCTGGAGGATAG-3';

R71rl - 5'-CCTGTCCACCTCGGCCCGCCTATC-3';

The primers E70f1 and R71r1 were introduced nucleotide polymorphisms, so that annealing was performed only on the alleles determining resistance to leukemia. The primers do not interact (not annealed) with alleles that cause increased sensitivity to the virus, and neutral alleles (Figure 1).

This allows to identify the presence of any potentially resistant leukemia alleles(*11, *23, *28=*7A) in a single reaction. This allele, not causing resistance to leukemia (sensitive and neutral)do not differentiate.

PCR was performed according to the following protocols in the reaction mixture of the following composition: 1×PCR buffer (16.6 mm (NH4)2SO4, 67.7 mm Tris-HCl, pH=8.8, 0.1% (v/v) Tween 20, 1.5 mm MgCl2), 200 mm dNTP, 10 pmol of each primer, 1 Unit Taq polymerase.

The verification of the effectiveness of passing PCR was diagnosed by the method of gel-electro is oresa, applying 5 µl of amplificate in 3% agarose gel electrophoresis were separated in 1×TAE buffer for 20 min at 100 V and were detected under ultraviolet light (UV).

Next, samples that showed electrophoresis 3 fragments of appropriate length (284, 233 and 93 BP)was subjected to another PCR Protocol (table 1.2), the effectiveness of the passage which was also checked using agarose gel electrophoresis.

The remaining 30 μl of amplification was mixed in a round bottom plastic 96-well-plates with 3 ál of sepharose (Streptavidin Sepharose™, 17-5113-03) and 57 μl of binding buffer (Binding Buffer, 40-0033), incubated at room temperature for 10 min, rapidly mixing on a shaker at 1400 rpm.

Using vacuum workstation (Vacuum Prep Workstation (220-240 V), 60-0207) associated with sepharose matrix PCR products were transferred to the preparation filters and then washed for 10 seconds in 70% ethanol, 0.2 M NaOH (DNA denaturation), 1× wash buffer (Wasching Buffer, 40-0035). After disconnecting vacuum samples 5-10 sec was suirable in the prepared thin-walled plastic 96-well plates (PSQ 96 Plate Low, 40-0010)containing in each well, 45 ál of Annealing Buffer (40-0036) in a mixture of 15 picomoles Sequeira primers selected to mutations in amino acids 70 and 71 of the gene BoLA-DRB3 cattle. As securitysage we have chosen primer, 5'-horse is which Machen Biotin, l30 bio 5'-BIOATCCTCTCTCTGCAGCACATTTCC-3', and developed an analysis scheme that allows simultaneous determination of the polymorphism of the amplified gene BoLA-DRB3 cattle in positions 2 codons 70 and 71.

The thus prepared sample was placed in a thermostat, incubated for 2 min at 90°C for hybridization Sequeira primers to single-stranded DNA-matrix, after cooled at room temperature for 10 minutes

The genodiagnostic conducted on automatic pyrosequencer PSQ™ 96MA SYSTEM (Pyrosequencing AB) using reagents from a set PSQ™ 96 SNP Reagent Kit 5×96 (40-0023) in real-time. Processing raw data persecutione studied SNP was performed using the software PSQ96MA SNP software v.2.0 (Figure 2).

So determined for each animal alleles summed up in a Microsoft Excel spreadsheet. The resulting matrix of genotypes served as a basis for statistical analysis of the results.

Example 2. The developed system was tested on 344 animals black and white breed (Table 3).

The survey revealed that 20,93% of animals are carriers of resistant alleles, while 4,07% are homozygous for these alleles. When diagnosing leukemia virus by PCR among all carriers of resistant alleles were detected only 6,94% of patients, while SRT is t be noted, among homozygotes carriers of the virus have been identified.

Among heterozygotes were identified 5 animals-carriers of the virus, that is to 8.62%.

All homozygotes and heterozygotes for sustainable alleles were REED-negative.

Using the developed system were also analyzed DNA samples from 30 of the bulls of breeds Holstein root (red-spotted, black and white, Ayrshire), published on 6 plempredpriyaty Russia (Table 4).

It was found that 13,33% of the animals are carriers of resistant alleles, while 6,67% were homozygote on these alleles. All animals were free from provirus leukemia.

The information obtained shows the feasibility of using the developed system for the characterization of the gene pool breeding animals.

The invention allows to reduce the costs of working time, labour and money for testing the resistance of cattle to leukemia by reducing the number of the analyzed nucleotide sequences.

The invention can be used for mass genotyping of animals resistant leukemia in BoLA-DRB3, breeding and commercial farms for the selection of animals in the nucleus.

Table 1
The PCR protocols
Bola 1 table 1.1Bola 2 psq table 1.2
The main solutionComponentsThe working solutionálThe main solutionComponentsThe working solutionál
H2O10,3H2O27,65
10×The buffer1,510×The buffer3,5
2 mmdNTP21,52 mmdNTP21,75
50 pmol/álBoa10 pmol0,250 pmol/álVOA bio10 pmol0,2
50 pmol/álBoa10 pmol0,250 pmol/álBoa10 pmol0,2
100 mmMgCl20,2 100 mmMgCl20,1
5 U/µlTaq1U0,15 U/µlTaq1U0,1
DNA1The product of the 1st PCR1,5
Finite volume15
Finite volume35
/td>
Bola 2 al.sp. Table 1.3
The main solutionComponentsThe working solutionál
H2O22,9
10×The buffer3,0
2 mmdNTP21,5
50 pmol/álBoa10 pmol0,2
50 pmol/álBoa10 pmol0,2
50 pmol/álE70fl10 pmol0,2
50 pmol/álR71rl10 pmol0,2
100 mmMgCl20,2
5 U/µlTaq1U0,1
The product of the 1st PCR1,5
Finite volume30

Table 2
Temperature-time mode Rea the Nations amplification
The first reaction amplificationThe second reaction amplification
TemperatureThe number of cyclesTimeTemperatureThe number of cyclesTime
95°C17 min95°C17 min
94°C101 min94°C301 min
60°C2 min52°C30 sec
72°C1 min72°C1 min
72°C17 min72°C17 min

table 3
The distribution of animals of black-motley breed depending on the genotype of the gene BoLA-DRB3
The genotype for the gene BoLA-DRB3The frequency of occurrence of genotype ERDiagnosis provirus PCRThe results of REID
+-+
n%n%n%n%
Resistant alleles (ER), including : 7220,9356,946793,0600
homozygotes 144,0700,0014100,0000
heterozygotes5816,8658,625391,3800
Other alleles27279,075620,5921679,4110of 3.56
Just herd344100,006117,7328382,2710of 3.56
Table 4
The genotypes of bulls gene BoLA-DRB3 contained in 6 plempredpriyaty Russia
The genotype for the gene BoLA-DRB3The frequency of occurrence of genotype ERDiagnosis provirus PCR
+-
n%n%n%
Resistant alleles (ER), including : 413,3300,004100,00
homozygotes2to 6.67 00,002100,00
heterozygotes2to 6.6700,002100,00
Other alleles2686,6700,0026100,00
Only30100,0000,0030100,00

Method for diagnosis of resistance of cattle to leukosis virus, including allele-specific Nested-PCR with primers selected for nucleotide sequences coding for amino acids in which Aziziyah 70-71 amino acid sequence, wherein the allele-specific primers 70fl - 5'-AGAAGGAGATCCTGGAGGATAG-3' and R71r1 - 5'-CCTGTCCACCTCGGCCCGCCTATC-3', select the part of the gene BoLA-DRB3, located on 23 chromosome (localization 23q21):

they interact only with nucleotide sequences encoding alleles*11, *23, *28=*7A, causing genetic resistance to leukemia in cattle and forth using securitysage primer Zond 70/71 5'-GCCCGGCTACACCTGT-3' define Homo - or heterozygosity individuals on these alleles, while the interaction of primers with alleles*11, *23, *28=*7A animals referred to as resistant leukemia, and in the absence of interaction with the same alleles, they can be defined as unstable to leukemia, so and to neutral.



 

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