Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, Salt Lake City, Utah 84108-1221, USA.
J Mol Diagn. 2011 Jul;13(4):446-51. doi: 10.1016/j.jmoldx.2011.03.008. Epub 2011 May 14.
Recent genome-wide association studies have identified two host single-nucleotide polymorphisms (SNPs) near the IL28B gene (rs12979860 C/T and rs8099917 T/G) that are associated with sustained virological response in patients infected with the hepatitis C virus. Herein, we describe a rapid multiplexed dual-color fluorescence resonance energy transfer (FRET) probe assay that accurately genotypes for both SNPs simultaneously. A single-nucleotide extension assay was also developed for verification of genotypes. Agreement (100%) was observed in genotype calls between the FRET and single-nucleotide extension methods for both SNPs, yielding 100% analytical sensitivity and specificity. By using the FRET assay, 443 samples of varying ethnic backgrounds were genotyped and six different compound genotypes (rs12979860/rs8099917) were detected in whites, Asians, Middle Easterners, Hispanics, and African Americans, at the following frequencies: CC/TT (39.2%, 78.9%, 40.0%, 33.9%, and 16.8%), CT/TT (20.8%, 0%, 40%, 9.3%, and 37.0%), TT/TT (2.4%, 0%, 0%, 3.4%, and 35.3%), CT/TG (24.0%, 19.7%, 20%, 39.8%, and 3.4%), TT/TG (8.0%, 1.4%, 0%, 3.4%, and 5.9%), and TT/GG (5.6%, 0%, 0%, 10.2%, and 1.7%), respectively. The multiplexed FRET assay can be used to effectively genotype for both SNPs in a single tube, with high analytical sensitivity and specificity.
最近的全基因组关联研究已经确定了两个位于 IL28B 基因附近的宿主单核苷酸多态性（SNP）（rs12979860C/T 和 rs8099917T/G），它们与丙型肝炎病毒感染患者的持续病毒学应答相关。在此，我们描述了一种快速的多重双色荧光共振能量转移（FRET）探针检测法，该方法可以同时准确地对这两个 SNP 进行基因分型。还开发了一种单核苷酸延伸检测法来验证基因型。FRET 和单核苷酸延伸方法在两种 SNP 的基因型判断中均具有 100%的一致性，从而产生了 100%的分析灵敏度和特异性。使用 FRET 检测法，对来自不同种族背景的 443 个样本进行了基因分型，在白人、亚洲人、中东人、西班牙裔和非裔美国人中发现了六种不同的复合基因型（rs12979860/rs8099917），其频率分别为：CC/TT（39.2%、78.9%、40.0%、33.9%和 16.8%）、CT/TT（20.8%、0%、40%、9.3%和 37.0%）、TT/TT（2.4%、0%、0%、3.4%和 35.3%）、CT/TG（24.0%、19.7%、20%、39.8%和 3.4%）、TT/TG（8.0%、1.4%、0%、3.4%和 5.9%）和 TT/GG（5.6%、0%、0%、10.2%和 1.7%）。该多重 FRET 检测法可用于在单个管中有效地对两个 SNP 进行基因分型，具有高分析灵敏度和特异性。
