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RU2017106872A - METHOD FOR SIMULTANEOUS GENODIAGNOSIS OF FOUR MUTANT CAPPA-CASEIN ALLELLES IN CATTLE CATTLE AND TEST SYSTEM FOR ITS IMPLEMENTATION - Google Patents

METHOD FOR SIMULTANEOUS GENODIAGNOSIS OF FOUR MUTANT CAPPA-CASEIN ALLELLES IN CATTLE CATTLE AND TEST SYSTEM FOR ITS IMPLEMENTATION Download PDF

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RU2017106872A
RU2017106872A RU2017106872A RU2017106872A RU2017106872A RU 2017106872 A RU2017106872 A RU 2017106872A RU 2017106872 A RU2017106872 A RU 2017106872A RU 2017106872 A RU2017106872 A RU 2017106872A RU 2017106872 A RU2017106872 A RU 2017106872A
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csn3
pcr
cattle
reaction
rtq1
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RU2017106872A
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RU2017106872A3 (en
RU2691995C2 (en
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Саида Нурбиевна Марзанова
Давудай Абдулсемедович Девришов
Яков Игоревич Алексеев
Нина Валерьевна Коновалова
Нурбий Сафарбиевич Марзанов
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Общество с ограниченной ответственностью "АгроВет"
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Publication of RU2017106872A3 publication Critical patent/RU2017106872A3/ru
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Claims (4)

1. Способ одновременной генодиагностики четырех (κ-CNA, κ-CNB, κ-CNC, κ-CNE) мутантных аллелей каппа-казеина у крупного рогатого скота включает выделение ДНК из биологического материала, постановку полимеразной цепной реакции в режиме реального времени, с использованием трех реакционных смесей, содержащей все необходимые реактивы для проведения ПЦР-РВ, разбавитель, Taq ДНК-полимеразу, три положительных и один отрицательный контрольный образец для каждой реакционной смеси, 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. The method for simultaneous gene diagnostics of four (κ-CN A , κ-CN B , κ-CN C , κ-CN E ) mutant kappa-casein alleles in cattle involves the isolation of DNA from biological material, the formulation of the polymerase chain reaction in real time time, using three reaction mixtures containing all the necessary reagents for PCR-RV, diluent, Taq DNA polymerase, three positive and one negative control sample for each reaction mixture, in preparation for the reaction, the required volume of components, based on the number of test samples plus 4, the reagents are mixed, then 20 μl of the prepared PCR mixture is added to the prepared tubes for PCR, 5 μl of control and test samples are added to each PCR tube, the tubes are placed in an amplifier, the primers are annealed stage of cycling at 64 ° C for 30 s with the number of amplification cycles equal to 40, analysis of the obtained data is carried out by comparing amplified sections of genes, the results are interpreted based on the presence or absence of intersection to fluorescence fluorescence with a threshold line set at an appropriate level. 2. Тест-система для осуществления способа по п. 1 методом постановки полимеразной цепной реакции в режиме реального времени, характеризующаяся тем, что включает реактивы в виде трех комплектов, каждый комплект состоит из компонентов (реакционная смесь, разбавитель, полимераза и 4 контрольных образца), которые необходимо смешать в нужном объеме непосредственно перед проведением исследования. ПЦР смесь, содержащую все необходимые реагенты для проведения ПЦР РВ, а именно 50 мМ KCl, 50 мМ TRIS-HCl, 250 нМ dNTP, 2,5 мМ MgCl2, праймеры - в концентрации 200 нМ, флуоресцентно-меченных олигонуклеотидных проб - в концентрации 100 нМ, имеющие следующие последовательности:2. The test system for implementing the method according to claim 1 by the method of setting the polymerase chain reaction in real time, characterized in that it includes reagents in the form of three sets, each set consists of components (reaction mixture, diluent, polymerase and 4 control samples) , which must be mixed in the right amount immediately before the study. PCR mixture containing all the necessary reagents for PCR RT, namely 50 mM KCl, 50 mM TRIS-HCl, 250 nM dNTP, 2.5 mM MgCl 2 , primers at a concentration of 200 nM, fluorescently-labeled oligonucleotide samples at a concentration 100 nM having the following sequences: CSN3_FCSN3_F tgtgctgagtaggtatcctagttatggtgtgctgagtaggtatcctagttatgg CSN3_RCSN3_R gcgttgtcttctttgatgtctccttaggcgttgtcttctttgatgtctccttag CSN3-HinfI-wt2CSN3-HinfI-wt2 (FAM)-tagctactctagaagattctccaga-(RTQ1)(FAM) -tagctactctagaagattctccaga- (RTQ1) CSN3-HinfI-m2CSN3-HinfI-m2 (R6G)-gctactctagaagcttctccagaa-(BHQ2)(R6G) -gctactctagaagcttctccccagaa- (BHQ2) CSN3-HaeIII-wtCSN3-HaeIII-wt (FAM)-agaagttattgagAgcccacct-(RTQ1)(FAM) -agaagttattgagAgcccacct- (RTQ1) CSN3-HaeIII-m2CSN3-HaeIII-m2 (R6G)-cagaagttattgagGgccc-(BHQ2)(R6G) -cagaagttattgagGgccc- (BHQ2) CSN3-MaeII-wtCSN3-MaeII-wt (FAM)-catggcacGtcacccaca-(RTQ1)(FAM) -catggcacGtcacccaca-- (RTQ1) CSN3-MaeII-mCSN3-MaeII-m (R6G)-tggcacAtcacccacacc-(BHQ2),(R6G) -tggcacAtcacccacacc- (BHQ2),
разбавитель, 2,5 ед. Taq ДНК-полимеразы.diluent, 2.5 units Taq DNA polymerase.
RU2017106872A 2017-03-02 2017-03-02 Method for simultaneous genodiagnostic of four mutant alleles of kappa-casein in cattle and test system for implementation thereof RU2691995C2 (en)

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RU2386700C1 (en) * 2008-11-18 2010-04-20 ООО "Лаборатория Изоген" Method of detemining a- and b-alleles of cattle kappa casein gene through tetra-primer pcr
RU2601151C2 (en) * 2015-02-11 2016-10-27 Общество с ограниченной ответственностью "АгроВет" Method of simultaneous genodiagnostic of two mutant alleles causing cvm and blad in cattle, and test system for it

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