ПЛР-діагностика <i>blad</i> великої рогатої худоби та особливості подвійного носійства спадкових хвороб
The study was conducted on 57 bull-sires of Ukrainian black and red and white Holstein dairy breeds and five regional bulls breeding center National companies in animal breeding "Ukrplempidpryyemstvo". Blood for the study were collected from the jugular vein into tubes with sodium citrate. DNA was extracted from lymphocytes using a kit of reagents "DNA Sorbo -In" For DNA amplification method used polymerase chain reaction (PCR) using Thermocyclers (termotsyklera programmable for amplification ).The method was repeated in the direct synthesis of DNA in vitro DNF using thermostable Taq-F DNA polymerase. Hydrolysis was performed by incubating amplified DNA with restriction Hae III (0,5 ml ) in a one-time buffer composition is specially optimized for particular restriction enzymes for 1.5 h at a temperature of + 65 ° C. Horizontal electrophoresis was performed in 5% agarose gels for 20 minutes. After electrophoresis, the gel stained with ethidium bromide solution (0.5 mg / ml) for 4-6 min, washed, transferred to transilyuminator, spent photographic documentation of results. For result analysis used computer program Gel Explorer. The same distribution of restriction fragments observed a similar pattern in most animals. However, three bulls distribution of restriction fragments was different, allowing the judge that they were carriers of the disease. A computer program Gel Explorer allows you to build profilohrams electrophoretic distribution of restriction fragments in agarose gels. These graphics are accompanied by an indication of the current measurement: I - intensity of luminescence (from 0 to 255 units.) And L - molecular weight in units For profilohrames heterozygous carriers were characterized by three peaks of luminescence intensity of 145, 133 and 145 units. The first and third peaks had molecular weights of 51 and 114 arbitrary units, indicating similar to the first case, separate the products of restriction, but the presence of a third peak with a molecular mass of 69 USD between evidence of differences in the structure of restriction sites of the first bull, typical for medium bulls leukocytes adhesion deficiency (BLAD). The analysis profilohrams match those visual inspection of the results of electrophoresis as in healthy animals displays two DNA fragments (45, 87 bp) in patients - three (45, 68, 87 bp).Of the 57 subjects bulls similar to the first case, the distribution of restriction fragments observed in three. It should be noted that these same animals were both heterozygous carriers of complex defect of the spine (CVM). At the time of this research result was unexpected and demanded an explanation of dual carriers of inherited disease. However, analysis of literature sources yielded conclusive answers to any questions. It was found that widespread among CVM holsteins was due to an outstanding bull-sires KM 1667366 Bell, who was also a carrier of other hereditary defect - leukocyte adhesion deficiency (BLAD). At the moment we know that from KM Bell in the U.S. alone had received more than 79000 daughters measured in performance and more than 1200 children evaluated for their daughters. The main source of the spread of a lethal gene in Ukraine are descendants of KM Bell 1667366 BL and PS Shake BL 1617421. Carriers of the mutant gene and not tested children found in lines Eleveyshn 1491007 - H.H. Starbuck 352790, P.F.A. Chif 1427381. These bulls have received the mutant allele for maternal ancestry. Analyzing pedigrees identified carriers (BLAD) included in the group of animals with a high probability of the presence of the defective allele ("risk"). Thus, three of the 57 bulls were heterozygous carriers bulls leukocytes adhesion deficiency (BLAD) and complex defect of the spine (CVM).Tested method allows accurate diagnosis Deficit bulls leukocytes adhesion deficiency (BLAD) in heterozygous carriers. For diagnostic bulls leukocytes adhesion deficiency (BLAD) in heterozygous carriers need to consider simultaneous carriage integrated defect of the spine (CVM).