Abstract

β-Lactoglobulin (BLG) is one of the prevalent whey protein in cattle. To date, several variants of bovine BLG have been found, but the most common are A and B, which differ from each other by SNPs rs109625649 and rs110066229. Numerous studies showed effects of A and B variants of BLG on milk yield, fat and protein content and cheese-making properties. To date, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), allele-specific polymerase chain reaction (ASPCR), PCR single-strand conformation polymorphism (PCR-SSCP) and high resolution melting (HRM) methods have been proposed for detection of A and B variants of bovine BLG. These methods involve multistep sample processing, which is an essential disadvantage in conducting large-scale cattle genotyping projects. This article describes a development of TaqMan PCR assay for detection of A and B variants (rs109625649) of bovine BLG. In this method a primer pair, initiating amplification of 101-bp fragment of BLG gene, and two allele-specific TaqMan probes are used. Identification of B and A variants of BLG is based on comparison of final fluorescence intensity of FAM and VIC dyes, respectively. The developed one-step method requires less time and is more suitable for large-scale genotyping of cattle compared to the commonly used PCR-RFLP.

摘要

β-乳球蛋白 (BLG) 是牛中普遍存在的乳清蛋白之一。迄今为止，已经发现了几种牛 BLG 变体，但最常见的是 A 和 B，它们在 SNP rs109625649 和 rs110066229 上有所不同。大量研究表明 BLG 的 A 和 B 变体对牛奶产量、脂肪和蛋白质含量以及奶酪制作特性的影响。迄今为止，已经提出了聚合酶链反应-限制性片段长度多态性（PCR-RFLP）、等位基因特异性聚合酶链反应（ASPCR）、PCR单链构象多态性（PCR-SSCP）和高分辨率熔解（HRM）方法。检测牛 BLG 的 A 和 B 变体。这些方法涉及多步样品处理，这是进行大规模牛基因分型项目的一个基本缺点。本文介绍了一种用于检测牛 BLG 的 A 和 B 变体 (rs109625649) 的 TaqMan PCR 检测方法。在该方法中，使用一对引物，开始扩增 BLG 基因的 101-bp 片段，并使用两个等位基因特异性 TaqMan 探针。BLG 的 B 和 A 变体的鉴定分别基于 FAM 和 VIC 染料的最终荧光强度的比较。与常用的 PCR-RFLP 相比，开发的一步法所需时间更短，更适合牛的大规模基因分型。