RU2015104459A - Method for the simultaneous genetic diagnosis of two mutant alleles causing CVM and BLAD in cattle and a test system for its implementation - Google Patents

Method for the simultaneous genetic diagnosis of two mutant alleles causing CVM and BLAD in cattle and a test system for its implementation Download PDF

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RU2015104459A
RU2015104459A RU2015104459A RU2015104459A RU2015104459A RU 2015104459 A RU2015104459 A RU 2015104459A RU 2015104459 A RU2015104459 A RU 2015104459A RU 2015104459 A RU2015104459 A RU 2015104459A RU 2015104459 A RU2015104459 A RU 2015104459A
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blad
cvm
pcr
reaction
bhq2
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RU2015104459A
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RU2601151C2 (en
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Саида Нурбиевна Марзанова
Давуд Абдулсемидович Девришов
Яков Игоревич Алексеев
Нина Валерьевна Коновалова
Нурбий Сафарбиевич Марзанов
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Общество с ограниченной ответственностью "АгроВет"
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

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Abstract

1. Способ одновременной генодиагностики двух мутантных аллелей, вызывающих CVM и BLAD у крупного рогатого скота, включает выделение ДНК из биологического материала, постановку полимеразной цепной реакции в режиме реального времени, с использованием реакционной смеси с CVM/BLAD, содержащейвсе необходимые реактивы для проведения ПЦР-РВ, разбавитель, Taq ДНК-полимеразу, три положительных и один отрицательный контрольный образец, при подготовке к проведению реакции рассчитывают необходимый объем компонентов, исходя из количества исследуемых образцов плюс 4, реактивы смешивают, затем в подготовленные для ПЦР пробирки вносят по 20 мкл приготовленной ПЦР-смеси,в каждую ПЦР пробирку добавляют по 5 мкл контрольных и исследуемых образцов, пробирки помещают в амплификатор, отжиг праймеров проходит на этапе циклирования при 64 °С в течение 30 с при числе циклов амплификации равном 40, анализ полученных данных проводят путем сравнения амплифицированных участков генов, результаты интерпретируют на основании наличия или отсутствия пересечения кривой флуоресценции с установленной на соответствующем уровне пороговой линией.2. Тест-система для осуществления способа по п. 1 методом постановки полимеразной цепной реакции в режиме реального времени, характеризующаяся тем, что включает реакционную смесьс CVM/BLAD, содержащую все необходимые реагенты для проведения ПЦР РВ, а именно 50 мМKCl, 50 мMTRIS-HCl, 250 нМdNTP, 2,5 мМMgCl, праймеры - в концентрации 200 нМ, аллель-специфические зонды - в концентрации 100 нМ, имеющие следующие последовательности:CVM_up - gattctcaagagcttaattctaaggaCVM_low - aagtaaaccccagcaaagccacCVM_Wt - (FAM) aggtctcatggcagttct-(BHQ1)CVM_m - (R6G) catggcatttctcacagcat-(BHQ2),BLAD_up - ttaggcagttgcgttcBLAD_low - acgttgacgaggtcatccacca,BLAD_Wt - (ROX) accccatcgacctgtacta-(BHQ1),BLAD_m (Cy5) ccatcggcctgtactacct-(BHQ2),разбавитель, 2,5 ед. Taq ДНК-полимеразы, 3 положительных контрольных образца, один отрицательный контрольный образец.1. A method for the simultaneous genetic diagnosis of two mutant alleles that cause CVM and BLAD in cattle, involves the extraction of DNA from biological material, the formulation of the polymerase chain reaction in real time, using the reaction mixture with CVM / BLAD containing all the necessary reagents for PCR PB, diluent, Taq DNA polymerase, three positive and one negative control sample, in preparation for the reaction, the required volume of components is calculated based on the number of samples studied Cts plus 4, the reagents are mixed, then 20 μl of the prepared PCR mixture are added to the prepared PCR tubes, 5 μl of control and test samples are added to each PCR tube, the tubes are placed in an amplifier, the primers are annealed at the cycling stage at 64 ° С within 30 s with the number of amplification cycles equal to 40, the analysis of the obtained data is carried out by comparing the amplified regions of the genes, the results are interpreted based on the presence or absence of the intersection of the fluorescence curve with the corresponding uyuschem level threshold liniey.2. The test system for implementing the method according to claim 1 by the method of formulation of the polymerase chain reaction in real time, characterized in that it includes a CVM / BLAD reaction mixture containing all the necessary reagents for PCR RV, namely 50 mMKCl, 50 mMTRIS-HCl, 250 nMdNTP, 2.5 mMMgCl, primers at a concentration of 200 nM, allele-specific probes at a concentration of 100 nM, having the following sequences: CVM_up - gattctcaagagcttaattctaaggaCVM_low - aagtaaaccccagcaaagccacCVM_Wt - (FAM) aggctctcgtctcgctctgtgtgtgtgtgtgtgtgtgtgtgtag (BHQ2), BLAD_up - ttaggcagttgcgttcBLAD_low - acgttgacgaggtcatccacca, BLAD_Wt - (ROX) accccatcgacctgtacta- (BHQ1), BLAD_m (Cy5 ) ccatcggcctgtactacct- (BHQ2), diluent, 2.5 units Taq DNA polymerase, 3 positive controls, one negative control.

Claims (2)

1. Способ одновременной генодиагностики двух мутантных аллелей, вызывающих CVM и BLAD у крупного рогатого скота, включает выделение ДНК из биологического материала, постановку полимеразной цепной реакции в режиме реального времени, с использованием реакционной смеси с CVM/BLAD, содержащейвсе необходимые реактивы для проведения ПЦР-РВ, разбавитель, Taq ДНК-полимеразу, три положительных и один отрицательный контрольный образец, при подготовке к проведению реакции рассчитывают необходимый объем компонентов, исходя из количества исследуемых образцов плюс 4, реактивы смешивают, затем в подготовленные для ПЦР пробирки вносят по 20 мкл приготовленной ПЦР-смеси,в каждую ПЦР пробирку добавляют по 5 мкл контрольных и исследуемых образцов, пробирки помещают в амплификатор, отжиг праймеров проходит на этапе циклирования при 64 °С в течение 30 с при числе циклов амплификации равном 40, анализ полученных данных проводят путем сравнения амплифицированных участков генов, результаты интерпретируют на основании наличия или отсутствия пересечения кривой флуоресценции с установленной на соответствующем уровне пороговой линией. 1. A method for the simultaneous genetic diagnosis of two mutant alleles that cause CVM and BLAD in cattle, involves the extraction of DNA from biological material, the formulation of the polymerase chain reaction in real time, using the reaction mixture with CVM / BLAD containing all the necessary reagents for PCR PB, diluent, Taq DNA polymerase, three positive and one negative control sample, in preparation for the reaction, the required volume of components is calculated based on the number of samples studied Cts plus 4, the reagents are mixed, then 20 μl of the prepared PCR mixture are added to the prepared PCR tubes, 5 μl of control and test samples are added to each PCR tube, the tubes are placed in an amplifier, the primers are annealed at the cycling stage at 64 ° С within 30 s with the number of amplification cycles equal to 40, the analysis of the obtained data is carried out by comparing the amplified regions of the genes, the results are interpreted based on the presence or absence of the intersection of the fluorescence curve with the corresponding uyuschem threshold line. 2. Тест-система для осуществления способа по п. 1 методом постановки полимеразной цепной реакции в режиме реального времени, характеризующаяся тем, что включает реакционную смесьс CVM/BLAD, содержащую все необходимые реагенты для проведения ПЦР РВ, а именно 50 мМKCl, 50 мMTRIS-HCl, 250 нМdNTP, 2,5 мМMgCl2, праймеры - в концентрации 200 нМ, аллель-специфические зонды - в концентрации 100 нМ, имеющие следующие последовательности:2. The test system for implementing the method according to claim 1 by the method of setting up the polymerase chain reaction in real time, characterized in that it includes a CVM / BLAD reaction mixture containing all the necessary reagents for PCR RV, namely 50 mMKCl, 50 mMTRIS- HCl, 250 nMdNTP, 2.5 mMMgCl 2 , primers at a concentration of 200 nM, allele-specific probes at a concentration of 100 nM, having the following sequences: CVM_up - gattctcaagagcttaattctaaggaCVM_up - gattctcaagagcttaattctaagga CVM_low - aagtaaaccccagcaaagccacCVM_low - aagtaaaccccagcaaagccac CVM_Wt - (FAM) aggtctcatggcagttct-(BHQ1)CVM_Wt - (FAM) aggtctcatggcagttct- (BHQ1) CVM_m - (R6G) catggcatttctcacagcat-(BHQ2),CVM_m - (R6G) catggcatttctcacagcat- (BHQ2), BLAD_up - ttaggcagttgcgttcBLAD_up - ttaggcagttgcgttc BLAD_low - acgttgacgaggtcatccacca,BLAD_low - acgttgacgaggtcatccacca, BLAD_Wt - (ROX) accccatcgacctgtacta-(BHQ1),BLAD_Wt - (ROX) accccatcgacctgtacta-- (BHQ1), BLAD_m (Cy5) ccatcggcctgtactacct-(BHQ2), BLAD_m (Cy5) ccatcggcctgtactacct- (BHQ2), разбавитель, 2,5 ед. Taq ДНК-полимеразы, 3 положительных контрольных образца, один отрицательный контрольный образец. diluent, 2.5 units Taq DNA polymerase, 3 positive controls, one negative control.
RU2015104459/10A 2015-02-11 2015-02-11 Method of simultaneous genodiagnostic of two mutant alleles causing cvm and blad in cattle, and test system for it RU2601151C2 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110272994A (en) * 2019-07-15 2019-09-24 中国医学科学院北京协和医院 Diagnose the gene mutation and its application of CVM

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2691995C2 (en) * 2017-03-02 2019-06-19 Общество с ограниченной ответственностью "АгроВет" Method for simultaneous genodiagnostic of four mutant alleles of kappa-casein in cattle and test system for implementation thereof
RU2731058C1 (en) * 2019-11-26 2020-08-28 Федеральное государственное бюджетное образовательное учреждение высшего образования "Костромская государственная сельскохозяйственная академия" Test system for detecting single nucleotide substitution (a→g) of position 383 of cd18 gene associated with deficit of lymphocyte adhesion in cows (blad), using specific oligonucleotide primers in polymerase chain reaction

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110272994A (en) * 2019-07-15 2019-09-24 中国医学科学院北京协和医院 Diagnose the gene mutation and its application of CVM
CN110272994B (en) * 2019-07-15 2021-01-26 中国医学科学院北京协和医院 Gene mutation diagnosis of CVM and application thereof

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