RU2691995C2 - Method for simultaneous genodiagnostic of four mutant alleles of kappa-casein in cattle and test system for implementation thereof - Google Patents

Abstract

FIELD: veterinary medicine.SUBSTANCE: group of inventions relates to molecular genetics and can be used in veterinary practice and animal husbandry for diagnosing four kappa-casein alleles. Disclosed is a method for simultaneous genodiagnostics of four mutant kappa-casein alleles in cattle, involving DNA recovery from biological material, polymerase chain reaction in real time, using three reaction mixtures containing 50 mM KCl, 50 mM TRIS-HCl, 250 nM dNTP, 2.5 mM MgCl, primers - in concentration of 200 nM, fluorescent-marked oligonucleotide samples - in concentration of 100 nM, 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 is calculated based on the number of analyzed samples plus 4, the reagents are mixed, then in prepared for PCR test tubes are introduced by 20 mcl of prepared PCR-mixture, in each PCR test tube is added by 5 mcl of control and analyzed samples, test tubes are placed in a thermocycler, annealing of primers passes at cycling step 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 the amplified gene sections, results are interpreted on the basis of presence or absence of intersection of the fluorescence curve with a threshold line installed at the corresponding level. Also disclosed is a test system for implementing said method.EFFECT: group of inventions enables diagnosing four alleles simultaneously; faster execution of PCR-PB; reduced labour intensity; possibility of monitoring accuracy of analysis.2 cl, 1 dwg, 4 tbl

Description

The invention relates to the field of molecular genetics and can be used in veterinary practice and animal science for the diagnosis of four important kappa-casein alleles (κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E ). The proposed method and test system for its implementation allow you to simultaneously identify four alleles in the kappa-casein locus using real-time PCR (PCR-RV) based on primers and allele-specific probes.

In cattle, 15 kappa-casein variants are known, however, due to laboratory possibilities, the CN ^A and CN ^B alleles were most often detected (Prinzenberg, EM et al :, 1999; Strzalkowska N. et al., 2002). It was established that the allele takes part in the formation of resistance to the temperature regime of milk proteins, reducing the coagulation time, better curving and the formation of micelles of various sizes that are most suitable for making cheese (Mao IL et al., 1992; Prinzenberg EM et al. 1999). The yield of cheese from milk of cows with the κ-CN ^BB genotype is 10% higher compared to the κ-CN ^AA variant. The presence of κ-CN ^B allele contributes not only to an increase in the yield of cheese and to an improvement in its quality, it also correlates with other valuable parameters of milk production (protein content in milk and milk production). The effect of the κ-CN ^B allele on the protein content in milk has been shown on many breeds, including the black-and-white Holsteinized and even the zebu x black-and-white hybrids. This situation can be explained by the phenomenon caused by the substitution of amino acids in the protein due to mutations in exon IV of the kappa-casein gene. Therefore, the selection of animals for κ-CN ^B allele is integrated into dairy cattle breeding programs of many countries of the world (Marziali AS, Ng-Kwai-Hang KF, 1986; Fox PF, 1992; Freyer G. et al., 1999; Lunden A., Afforselles J., 2000; Amerkhanov XA, Marzanov HC, 1999; Marzanov HC et al., 2010; 2014).

In the Russian Federation, breeding animals are tested for carriage of only two kappa-casein alleles (κ-CN ^A , κ-CN ^B ), and the results are mandatory, along with blood groups and mutant alleles that cause hereditary diseases, to be regularly published in catalogs according to breeding to bulls (Shapochkin V.V., Eskin G.V., 2004; Savenko N.A. et al., 2007; Eskin G.V. et al., 2010; 2014-2015).

In the open press there are a number of works in which not only diagnostic methods are proposed, but it is also shown that the quality of milk from animals with κ-CN ^B allele is better than with κ-CN ^A (Rahali V., Menard JL, 1991), κ -CN ^C (Macheboeuf D. et al., 1993) and κ-CN ^E (Gravert H. O. et al., 1991). Knowledge of point mutations that distinguish these alleles allowed us to develop PCR-RFLP methods for their detection for κ-CN ^A and κ-CN ^B (Denicourt D. et al., 1990; Medrano JF, Aguilar-Cordova E., 1990; Zadworny D., Kuhnlein U., 1990), κ-CN ^C (Schlee P., Rotmann O., 1992) and κ-CN ^E (Schlieben S. et al., 1991). However, the diagnosis of their separate identification takes a long time. Orita M. et al. (1989) published, as they believed at the time, a simple method for determining DNA mutations and called it the “PCR-single strand conformation polymorphism, PCR-SSCP) method. The method allowed to detect the presence of nucleotide substitutions, deletions and insertions throughout the entire length of the amplified product. The PCR-SSCP method is as follows: the amplification product is denatured and the resulting single-stranded molecules are separated using polyacrylamide gel electrophoresis. Any, even a single nucleotide change leads to a change in the charge and, as a consequence, a change in the conformation of the single-stranded fragment and its electrophoretic mobility in the gel, which makes it possible to identify polymorphic variants. However, this method does not allow to determine exactly what changes have occurred? Therefore, the PCR-SSCP method is often carried out together with subsequent sequencing.

Thus, in molecular genetics, there is an obvious need to develop a highly sensitive, specific and fast method for diagnosing the most frequently encountered four kappa-casein alleles (κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E ), as well as test systems for its implementation, which may be applicable for various production purposes, in particular breeding practice. In addition, due to ignorance of these issues, the purchased breeding material is not tested for κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E alleles, as a result of which Russian cattle breeds are clogged with undesirable kappa genotypes casein

A genetic test is known from the technical level to identify two kappa-casein alleles (κ-CN ^A , κ-CN ^B ) of cattle (patent for invention No. 2386700 published on April 20, 2010). This invention is taken as the closest analogue.

A distinctive feature of the claimed invention from the identified analog is the ability to immediately identify the four most common alleles (κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E ), i.e. There is a simultaneous determination of four variants by real-time PCR (PCR-RV), using primers and allele-specific probes, knowing the points of mutations, and the approach of detection is fundamentally different. In addition, according to the existing patent, it is possible to identify only two alleles (κ-CN ^A , κ-CN ^B ), which is not enough in modern conditions for assessing the technological capabilities of the animal, because under κ-CN ^A allele virtually all of its known kappa-casein variants are hidden. As an example, we present the results of certification of the breeding bull Smile 91941 of the Anglers breed for kappa-casein. When diallel (κ-CN ^A , κ-CN ^B ) was analyzed, he would have the κ-CN ^AA genotype, while using the method we proposed, in fact, he has κ-CN ^AC heterozygote. Thus, the κ-CN ^A allele seems to hide the clogging of herds and cattle with other derivatives, in particular κ-CK ^C and κ-CN ^E , which are the most common and worsening of the quality of milk for cheese use in diluted dairy cattle.

In the claimed invention, polymorphism is identified in the kappa-casein locus using PCR-RV and TaqMan probes. In the closest analogue, kappa-casein is diagnosed using an electrophoretic detection method, which is a fundamentally different method for diagnosing this polymorphism and takes more time, necessary equipment and reagents.

The aim of the present invention is to create a method for the simultaneous diagnosis of four alleles (κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E ) using real-time PCR (PCR-RV) and the development of a test system for its implementation using sets of oligonucleotide primers and probes having specific sequences.

The present invention presents a method for the simultaneous diagnosis of four alleles in different cattle breeds. The test system was developed for the certification of all sex and age groups of animals: sires, bykovoprepayuschih groups of cows, maintenance of young animals, as well as semen from spermobank and embryos.

The technical result of the claimed invention is the ability to diagnose simultaneously four alleles (κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E ); reduction of the time taken to complete the real-time polymerase chain reaction (PCR-RV) (a result was obtained after 3 hours); not labor intensive; the ability to perform repetitions; to monitor the correctness of the study.

This technical result is achieved by carrying out the isolation of DNA from biological material, setting the polymerase chain reaction in real time, using reagents. Reagents in the form of three sets. Each kit consists of components (reaction mixture, diluent and polymerase), which must be mixed in the right amount just before the study.

In preparation for the reaction, the required volume of components is calculated, based on the number of samples studied plus 4, of which three are positive control samples and one negative control sample for each reaction mixture. The reagents are mixed, then 20 μl of the prepared PCR mixture are added to the tubes prepared for PNR, 5 μl of the control and test samples are added to each PNR tube. The tubes are placed in the amplifier. Primers are annealed at the cycling stage at 64 ° C for 30 s with the number of amplification cycles equal to 40. The obtained data is analyzed by comparing the amplified regions of the genes, the results are interpreted based on the presence or absence of the fluorescence curve intersection with the threshold line set at the appropriate level.

The test system contains 3 reaction mixtures (50 mM KCl, 50 mM TRIS-HCl, 250 nM dNTP, 2.5 mM MgCl ₂ , primers at a concentration of 200 nM, allele-specific probes at a concentration of 100 nM), the diluent is deionized water 1 ml, 2.5 units. Taq DNA polymerases, as well as 3 positive control samples, one negative control sample for each reaction mixture. Primers and fluorescently-labeled oligonucleotide samples have the following sequences:

The optimal composition of the reaction mixture for carrying out the real-time polymerase chain reaction was experimentally established, the combination of primers, the necessary and sufficient ratio of the components of the reaction mixture were selected, and the mode of PCR formulation was determined, which is important during PCR analysis. The system is designed to conduct 100 reactions with a volume of 25 μl (20 μl of the reaction mixture + 5 μl of the sample under study).

Selection of primers and allele-specific probes (Table 1) was performed using the Oligo 6.0 application software package based on the analysis of the nucleotide sequences of the kappa-casein locus published in the database of the National Center for Biotechnology Information (NCBI). ).

The source for the diagnosis is pre-isolated DNA from samples of biological material (whole blood, sperm, embryos, milk or skin).

DNA isolation was carried out by the sorbent method (DNA-sorb-V, FBUN Central Research Institute of Epidemiology, Rospotrebnadzor, Moscow). Next, the isolated DNA was used in the reaction: 5 μl of the sample was added to the PCR reaction mixture.

The reaction mixture for PCR analysis has the following composition: 50 mM KCl, 50 mM TRIS-HCl, 25 mM dNTP, 250 mM MgCl ₂ , 2.5 units. Taq DNA polymerases, primers at a concentration of 200 nM, fluorescent probes at a concentration of 100 nM, while the primers and fluorescently labeled oligonucleotide samples have certain specific sequences (Table 1).

Preparation for carrying out the reaction was carried out as follows.

The required volume of the components was calculated based on the number of samples tested plus 4 (three positive control samples and one negative control sample) for each of the three reaction mixtures. A set of components for the reaction are presented in table 2.

Note: * - contains all the necessary components for PCR-RV. Storage conditions: №1-3, at a temperature of -16-20 ° С

The components were thawed, mixed and centrifuged. Preparing a mixture of components, adding them in the order indicated in table 3.

PCR tubes were labeled. In the tubes prepared for PCR, 20 μl of the mixture of components were added. Then 5 μl of the control and test samples were added to each PCR tube in accordance with the marking, mixed by repeated pipetting, and the lid was tightly closed. The tubes were placed in the amplifier, then the instrument was programmed.

Reaction Profile:

1. Keeping temperature 94 ° С - 3 min, one repetition.

2. Cycling 1 (without detection)

94 ° C - 20 sec

61 ° C - 20 sec

64 ° C - 30 sec

The cycle was repeated 10 times.

3. Cycling 2 (with detection)

94 ° C - 20 sec

61 ° C - 20 sec

64 ° С- 30 sec. - detection by channel: Green, Yellow

The cycle was repeated 30 times.

In the process of cycling, primer annealing and synthesis of a new DNA chain occurred at a temperature of 64 ° C. During the first cycling, no detection was performed, since the data are not representative. In the second cycle, 30 repetitions were performed, and stable data were obtained as a result, which are final in terms of the diagnosis of genotypes.

The creation of the PCR-RV template, as well as the analysis of the research results, was carried out using the Rotor-Gene 6000 1.8 software for the Rotor Gene Q instrument (Corbett Research, Australia).

The result of the analysis is the determination of the combination of four alleles of the kappa-casein gene (κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E ) in cattle. Interpretation of the results of the analysis of the studied samples was performed in accordance with Table 4:

Note: PC1, PC2, PC3-reaction mixtures 1, 2, 3, containing fluorescently labeled oligonucleotide samples with the following nitrogenous bases: adenine (A), cytosine (C), guanine (G), used to diagnose genotypes and alleles in the locus kappa casein; Green and Yellow fluorescence channels in the Rotor Gene Q device (Corbett Research, Australia) by which genotypes and alleles are determined at the kappa-casein locus

For a reliable analysis result, the following conditions must be met:

1. The reaction result for PKO1 should be positive in the Green channel, negative in the Yellow channel.

2. The result of the reaction for PKO2 should be positive in the Green and Yellow channels.

3. The reaction result for PKO3 should be positive in the Yellow channel, negative in the Green channel.

4. The reaction result for the Negative Control should be negative in both channels.

In the case of obtaining positive results for the EYE on any of the detection channels, the results of the study are considered invalid. It is necessary to take measures to identify and eliminate the source of contamination and to re-analyze.

The result of the analysis for a sample is considered undefined if the reaction through the Green and Yellow channels is negative. In this case, the sample must be retested.

In the claimed invention, the technology of differentiation of alleles using oligonucleotide samples is used. When using fluorescently labeled oligonucleotide probes, an approach based on the difference in the efficiency of hybridization of allele-specific probes on each PCR cycle is used to distinguish between single nucleotide polymorphisms (SNPs). A pair of hybridization samples for each reaction mixture, specific for each allelic variant, labeled with different fluorophores and destroyed during PCR using 5 - exonuclease activity of Taq - polymerase is used. The detection of each of the alleles is based on the difference in the efficiency of hybridization of the sample on each cycle of PCR with a fully complementary sample by the allele and allele with the replacement of one nucleotide. Since only hybridized samples are destroyed by the enzyme, signal accumulation is proportional to the efficiency of hybridization. The difference in the efficiency of hybridization of samples is very small, however, this small difference accumulates from cycle to cycle and, as a result, allows to reliably distinguish alleles.

The results are interpreted based on the presence or absence of the intersection of the fluorescence curve with the threshold line set at the appropriate level. Sample No. 1 of κ-CN ^{AC is} considered as an example (Table 4, Fig. 1).

The result of the reaction are fluorescent signal accumulation curves that cross the threshold line. For PC1, there are two curves in the figure that cross the threshold line, which corresponds to the AC heterozygote. For PC2, there is one curve in the figure that crosses the threshold line, which corresponds to the wild type or AA homozygote. For PC3, there are two curves in the figure that cross the threshold line, which corresponds to the heterozygote GA. The set of results for the three reaction mixtures allows to conclude that the test sample belongs to the genotype K-CN ^AC .

The main advantages of the claimed invention are:

1. Simultaneous certification of elite animals of various cattle breeds for four alleles (κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E ) using the declared test system will allow to purposefully form an allele fund of manufacturing bulls reproducing groups of cows, rearing youngsters, as well as accumulating breeding material (seed bank and embryos).

2. The presence of a genetic passport for four kappa-casein alleles (κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E ), as well as a test system for its implementation, will allow to effectively carry out breeding work in breeding herds of cattle of the Russian Federation.

3. When bringing breeding material to the Russian Federation from abroad, it is necessary to have a genetic passport for four kappa-casein alleles (κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E ). In order to confirm the genotype for kappa-casein, purchased animals after quarantine must be re-certified.

Literature:

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Claims (5)

1. Method for simultaneous genodiagnosis of four mutant kappa-casein alleles κ-CN ^A , κ-CN ^B , κ-CN ^C , κ-CN ^E in cattle includes the isolation of DNA from biological material, the production of real-time polymerase chain reaction, using three reaction mixtures containing 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 of 100 nM, having the following sequences: CSN3_F tgtgctgagtaggtatcctagttatgg CSN3_R gcgttgtcttctttgatgtctccttag CSN3-HinfI-wt2 (FAM) -tagctactctagaagattctccaga- (RTQ1) CSN3-HinfI-m2 (R6G) -gctactctagaagcttctccagaa- (BHQ2) CSN3-HaeIII-wt (FAM) -agaagttattgagAgcccacct- (RTQ1) CSN3-HaeIII-m2 (R6G) -cagaagttattgagGgccc- (BHQ2) CSN3-MaeII-wt (FAM) -catggcacGtcacccaca- (RTQ1) CSN3-MaeII-m (R6G) -tggcacAtcacccacacc- (BHQ2), 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 is calculated based on the number of test samples plus 4, the reagents are mixed, then 20 μl are added to the tubes prepared for PNR prepared PCR mixture, 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 ° C for 30 s at h follows amplification cycles of 40, data analysis is performed by comparing portions of the amplified genes, the results are interpreted on the basis of the presence or absence of fluorescence curve intersection installed at an appropriate level threshold line. 2. A test system for implementing the method according to claim 1 using a real-time polymerase chain reaction method, characterized in that it includes reagents in the form of three sets, each of which consists of a reaction mixture, a diluent, 2.5 units. Taq DNA polymerases and 3 positive control samples and one negative control sample for each reaction mixture, which must be mixed immediately prior to the study, where the reaction mixture contains all the necessary reagents for carrying out the RT-PCR, 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 of 100 nM, having the following sequences: CSN3_F tgtgctgagtaggtatcctagttatgg CSN3_R gcgttgtcttctttgatgtctccttag CSN3-HinfI-wt2 (FAM) -tagctactctagaagattctccaga- (RTQ1) CSN3-HinfI-m2 (R6G) -gctactctagaagcttctccagaa- (BHQ2) CSN3-HaeIII-wt (FAM) -agaagttattgagAgcccacct- (RTQ1) CSN3-HaeIII-m2 (R6G) -cagaagttattgagGgccc- (BHQ2) CSN3-MaeII-wt (FAM) -catggcacGtcacccaca- (RTQ1) CSN3-MaeII-m (R6G) -tggcacAtcacccacacc- (BHQ2).

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