Alyethodi Rafeeque R, Singh Umesh, Kumar Sushil, Deb Rajib, Alex Rani, Sharma Sheetal, Sengar Gyanendra S, Prakash B
ICAR-Central Institute for Research on Cattle, Grass Farm Road, Meerut Cantt, Meerut, UP 250001 India.
Springerplus. 2016 Aug 30;5(1):1442. doi: 10.1186/s40064-016-3148-7. eCollection 2016.
Fast and economical means of assaying SNP's are important in diagnostic assays, especially when a large number of animals have to be screened for a genetic disease. This study was aimed at the development of a fast and economical screening assay for bovine leukocyte adhesion deficiency (BLAD) which is an important genetic disease of cattle industry. Four primers were designed where the outer primers amplify a 354 bp amplicon of CD18 gene carrying the polymorphism responsible for BLAD. The specifically designed inner primers in conjunction with the modified reaction mixture and cyclic conditions ensured amplification of either of wild or mutated alleles. Together with outer primers, the inner primers generated typical banding pattern in agarose gel which discriminated the normal animal against the carrier. We successfully used this protocol in 200 bulls for genotyping the BLAD allele which confirmed by sequencing, showing a cent percentage concordance. With the developed assay the need for restriction digestion or use of costly equipment viz. real time PCR was eliminated. This genotyping assay ensured fast and economical genotyping and could be adopted in every laboratory with a minimum equipment requirement of thermocycler and gel documentation system.
快速且经济的单核苷酸多态性（SNP）检测方法在诊断检测中至关重要，尤其是在需要对大量动物进行遗传疾病筛查时。本研究旨在开发一种快速且经济的牛白细胞黏附缺陷症（BLAD）筛查检测方法，BLAD是养牛业一种重要的遗传疾病。设计了四条引物，其中外部引物扩增携带导致BLAD的多态性的CD18基因的354 bp扩增子。专门设计的内部引物与改良的反应混合物及循环条件相结合，确保野生型或突变型等位基因中的任何一个都能被扩增。内部引物与外部引物一起在琼脂糖凝胶中产生典型的条带模式，可区分正常动物和携带者。我们成功地将该方案用于200头公牛的BLAD等位基因基因分型，测序结果证实了该方法，显示出百分之百的一致性。利用所开发的检测方法，无需进行限制性酶切消化或使用昂贵的设备，即实时PCR。这种基因分型检测方法确保了快速且经济的基因分型，并且每个实验室只需具备最低限度的设备要求，即热循环仪和凝胶成像系统，就可以采用。
