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小鼠粪便样本中16S拷贝数的定量检测   

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摘要:16S rRNA基因和宏基因组测序技术是研究肠道菌群结构与功能的重要手段,由此可以获取微生物群落的结构和功能组成信息;然而,二者得到的数据均为相对丰度类型,大大削弱了不同样品间的可比性,也难以揭示生物量水平的菌群结构和功能变化。本文旨在运用实时荧光定量PCR技术 (Real-time PCR) 技术对小鼠粪便中的16S rRNA基因进行定量检测,为准确分析肠道菌群的结构提供技术参考。

关键词: 16S rRNA基因, 绝对定量, 肠道菌群

材料与试剂

试剂:

  1. 高保真酶 (PrimeSTAR, TAKARA, catalog number: R045A)
  2. TB Green酶 (TAKARA, catalog number: RR820A)
  3. Omega胶回收试剂盒 (Omega, catalog number: D2500-01)
  4. 琼脂糖
  5. 引物
    (27F-AGAGTTTGATCCTGGCTCAG, 1541R-AAGGAGGTGATCCAGCCGCA; 331F-TCCTACGGGAGGCAGCAGT, 797R-GGACTACCAGGGTATCTAATCCTGTT) (Jian等,2016)

仪器设备

  1. Nanodrop 2000
  2. 电泳仪
  3. 普通PCR仪 (Bio-read)
  4. 荧光定量PCR仪 (Bio-read)

实验步骤

一、全长16S rRNA基因标准品制备

  1. 全长16S rRNA基因扩增

    PCR扩增体系:


    PCR扩增程序 (30个循环):


  2. 全长16S rRNA基因扩增产物回收
    按照胶回收试剂盒说明书,对PCR扩增产物进行回收,并利用Nanodrop对其浓度进行测定。
  3. 全长16S rRNA基因扩增产物拷贝数确定
    举例:Nanodrop测得胶回收的16S片段的浓度为182 ng/μl,由于1,500 bp的16S核酸的分子量为5.1 x 105 ng/nmol (https://www.genscript.com/conversion.html),所以标准品中含有3.57 x 10-4 nmol分子,根据阿伏伽德罗常数计算1 μl标准品中含有2.14 x 1011个拷贝。
  4. 全长16S rRNA基因扩增产物梯度稀释
    将标准品做10倍系列稀释,使其形成109-104拷贝/μl。

二、16S rRNA基因拷贝数标准曲线的制备
  1. 全长16S rRNA基因梯度拷贝数标准品的定量PCR检测

    Real-time PCR扩增体系:


    PCR扩增程序 (40个循环):


  2. 构建16S rRNA基因梯度拷贝数标准曲线
    以不同拷贝数的阳性模板的对数为横坐标,以PCR反应过程中到达荧光阈值的初始循环数 (Ct) 为纵坐标得到标准曲线,为待测样品的定量提供了定量检测的参照标准。


三、小鼠粪便DNA待测样品中16S rRNA基因拷贝数的定量检测

对小鼠粪便DNA待测样品,按照“全长16S rRNA基因梯度拷贝数标准品的定量PCR检测”中的程序与条件,进行real-time PCR检测。依据建立的标准曲线,计算出待测样品中的16S rRNA基因拷贝数。

结果与分析

图1展示了溶解曲线、标准曲线和待测样品的检测结果,溶解曲线结果显示扩增片段的单一特异性,标准曲线的相关性表明该方法对于检测不同16S rRNA基因拷贝数的样本具有很好的线性关系。依据标准曲线方程,Ct值为8的小鼠粪便DNA样品中含有的16S rRNA基因拷贝数为10^ (-0.2819 x 8 + 10.682) = 2.67 × 108个。


图1. RT-PCR方法检测粪便样本中微生物16S rRNA基因拷贝数熔解曲线 (A-B) 和标准曲线 (C)

致谢

本文得到青年千人人才计划资助。

参考文献

  1.  Jian, C., Luukkonen, P., Yki-Jarvinen, H., Salonen, A. and Korpela, K. (2020). Quantitative PCR provides a simple and accessible method for quantitative microbiota profiling. PLoS One 15(1): e0227285.
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:刘红宾, 吕青青, 戴磊. (2020). 小鼠粪便样本中16S拷贝数的定量检测. Bio-101: e2003368. DOI: 10.21769/BioProtoc.2003368.
How to cite: Liu, H. B., Lv, Q. Q. and Dai, L. (2020). Quantitative Analysis of 16S rRNA Gene Copies in Mouse Fecal Sample. Bio-101: e2003368. DOI: 10.21769/BioProtoc.2003368.
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