双重荧光实时PCR法鉴定SRBⅠ基因敲除小鼠

Development of a duplex fluorescence RT-PCR assay for identifying SRBⅠgene knockout mice

目的建立一种双重荧光实时PCR鉴定B族Ⅰ型清道夫受体(SRBⅠ)基因敲除小鼠的方法。方法提取小鼠尾尖DNA,应用自行设计的鉴定野生型和敲除型SRBⅠ基因的引物和探针,经PCR扩增后,在FAM通道及CY5通道判断小鼠基因型。同时应用基因测序技术对结果进行验证,并构建质粒标准品分析该方法的灵敏度和重复性。结果仅在FAM通道出现典型S型扩增曲线的为SRBⅠ基因野生型小鼠,仅在CY5通道出现典型S型扩增曲线的为SRBⅠ基因敲除型小鼠,在两个通道都出现典型S型扩增曲线的为SRBⅠ基因杂合型小鼠。结果与DNA测序法吻合,检测野生型和突变型的灵敏度均达4×101拷贝/μL。结论新方法简单、快速、准确,适用于分型SRBⅠ基因敲除小鼠。

Objective To develop a duplex fluorescence RT-PCR assay for detection of scavenger receptor class B,typeⅠ（SRBⅠ） knockout mice. Methods Primers and probes were designed according to knockout region of SRBⅠ geneand related substituted sequence. DNA samples were extracted from tails of mice and performed amplification using real-time PCR. SRBⅠ genotypes of mice were analyzed according to amplification curves of FAM and CY5 channels. Finally, thesensitivity of the method was detected and the accuracy was verified by the direct sequencing. Results The homozygousSRBⅠ wild genotype showed an amplification curve only in FAM channel. When the homozygous SRBⅠ knockout genotypewas present, the typical S amplification curve appeared only in the CY5 channel. Heterozygous genotype showed two typicalS amplification curves in both FAM and CY5 channels, respectively. The results showed that the sensitivity reached 4×10^1copies/μL, and there was complete concordance between this method and direct DNA sequencing. Conclusion The newmethod is simple, rapid and accurate, which is suitable for genotyping SRBⅠ knockout mice.