天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (7): 732-734.doi: 10.11958/j.issn.0253-9896.2015.07.008

• 实验研究 • 上一篇    下一篇

双重荧光实时 PCR 法鉴定 SRBⅠ基因敲除小鼠

潘丽莉，郑璐，张俊，于洋，姚霜，喻妙梅，冯悦华，罗光华   

  1. 苏州大学附属第三医院综合实验室， 常州市个性化诊疗高技术研究重点实验室 （邮编213003）
  • 收稿日期:2015-01-21 修回日期:2015-03-03 出版日期:2015-07-15 发布日期:2015-07-15
  • 通讯作者: 罗光华 E-mail: shineroar@163.com E-mail:shineroar@163.com
  • 作者简介:潘丽莉 （1986）， 女， 医学学士， 主要从事疾病分子生物学诊断的研究
  • 基金资助:
    国家自然科学基金资助项目 （81201352）； 江苏省自然科学基金资助项目 （BK2012154）

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

PAN Lili, ZHENG Lu, ZHANG Jun, YU Yang, YAO Shuang, YU Miaomei, FENG Yuehua, LUO Guanghua   

  1. Comprehensive Laboratory， The Third Affiliated Hospital of Soochow University, Changzhou Key Lab of Individualized Diagnosis and Treatment Associated with High Technology Research, Changzhou 213003， China
  • Received:2015-01-21 Revised:2015-03-03 Published:2015-07-15 Online:2015-07-15
  • Contact: LUO Guanghua E-mail: shineroar@163.com E-mail:shineroar@163.com

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摘要： 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。
 

关键词: 抗原, CD36, 清道夫受体 BⅠ, 双重荧光实时 PCR, 基因敲除

Abstract: 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 gene and related substituted sequence. DNA samples were extracted from tails of mice and performed amplification using realtime PCR. SRBⅠ genotypes of mice were analyzed according to amplification curves of FAM and CY5 channels. Finally, the sensitivity of the method was detected and the accuracy was verified by the direct sequencing. Results The homozygous SRBⅠ wild genotype showed an amplification curve only in FAM channel. When the homozygous SRBⅠ knockout genotype was present, the typical S amplification curve appeared only in the CY5 channel. Heterozygous genotype showed two typical S amplification curves in both FAM and CY5 channels, respectively. The results showed that the sensitivity reached 4×101 copies/μL, and there was complete concordance between this method and direct DNA sequencing. Conclusion The new method is simple, rapid and accurate, which is suitable for genotyping SRBⅠ knockout mice.

Key words: antigens,CD36, scavenger receptors-BⅠ, dual fluorescence real-time PCR, gene knockout