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中华实验和临床感染病杂志(电子版) ›› 2018, Vol. 12 ›› Issue (02) : 193 -197. doi: 10.3877/cma.j.issn.1674-1358.2018.02.019

所属专题: 文献

基础论著

基于TaqMan-MGB探针荧光定量PCR技术检定细菌和人基因组拷贝数比值方法的建立
潘晓微1, 王贤军2,(), 刘云惠3, 杨宁敏3   
  1. 1. 325200 瑞安市,瑞安市中医院检验科
    2. 310006 杭州市,杭州市第一人民医院检验科
    3. 518000 深圳市,深圳致远精准医疗有限公司
  • 收稿日期:2017-04-07 出版日期:2018-04-15
  • 通信作者: 王贤军
  • 基金资助:
    浙江省自然科学基金(No. LY13H190003)

Establishment of method for determining the copy number ratio between bacterial and human genome based on TaqMan-MGB fluorescent quantitative PCR

Xiaowei Pan1, Xianjun Wang2,(), Yunhui Liu3, Ningmin Yang3   

  1. 1. Clinical Laboratory, Ruian Hospital of Traditional Chinese Medicine, Ruian 325200, China
    2. Clinical Laboratory, Hangzhou The First People’s Hospital, Hangzhou 518000, China
    3. Shenzhen Zhiyuan Precision Medical Co., Ltd., Shenzhen 518000, China
  • Received:2017-04-07 Published:2018-04-15
  • Corresponding author: Xianjun Wang
  • About author:
    Corresponding author: Wang Xianjun, Email:
引用本文:

潘晓微, 王贤军, 刘云惠, 杨宁敏. 基于TaqMan-MGB探针荧光定量PCR技术检定细菌和人基因组拷贝数比值方法的建立[J]. 中华实验和临床感染病杂志(电子版), 2018, 12(02): 193-197.

Xiaowei Pan, Xianjun Wang, Yunhui Liu, Ningmin Yang. Establishment of method for determining the copy number ratio between bacterial and human genome based on TaqMan-MGB fluorescent quantitative PCR[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2018, 12(02): 193-197.

目的

建立一种可以检测细菌和人基因组拷贝数比值的方法。

方法

分别设计细菌和人基因组特异性引物及通用探针。以大肠埃希菌和幽门螺杆菌混合菌液及人全血样本模拟混合样本。采用TaqMan-MGB荧光PCR方法对不同稀释浓度的混合样本进行定量分析。通过比较实测比值(Ct值换算成拷贝数的比值)与理论比值,评估本研究方法的可行性和准确性。

结果

经变异系数法(CV)分析,6个不同浓度样本(DB、DB2、DB3、DH1、DH2和DH3)的CV值依次为4.48%、1.57%、4.67%、1.55%、0.49%和1.29%。9组模拟混合样本中,实测比值(DB/DH拷贝数)和理论比值的误差均小于0.1,且两组数值相关性极显著(r = 1.000,P < 0.001)。

结论

本研究建立的基于TaqMan-MGB探针荧光定量PCR技术检定细菌和人基因组拷贝数比值的方法重复性和准确度良好,可用于细菌和人类组织样本基因组拷贝数比例的检定。

Objective

To establish a method for determining the copy number ratio of bacterial and human genome.

Methods

The specific primers and universal probes were designed for bacterial and human gene, respectively. The mixed samples consisted of E. coli, Helicobacter pylori and human blood and were quantified by TaqMan-MGB fluorescent PCR. The feasibility and accuracy were evaluated by comparing the measured ratio (the ratio of the Ct values converted into the copy number) and the theoretical ratio.

Results

The co-efficient of variation (CV) values of six samples (DB1, DB2, DB3, DH1, DH2 and DH3) were 4.48%, 1.57%, 4.67%, 1.55% , 0.49% and 1.29% by CV analysis, respectively. In the nine mixed samples, the error was less than 0.1 for the measured ratio (DB/DH) and the theoretical ratio, in which the correlation was significant between the two groups (r = 1.000, P < 0.001).

Conclusions

The method for determining the copy number ratio between bacterial and human genome based on TaqMan-MGB fluorescent quantitative PCR was reproducible and accurate, and could be used to determine the copy number ratio between bacteria and human genome.

表1 引物和探针序列
图1 DB扩增曲线和标准曲线
图2 DH扩增曲线和标准曲线
表2 拷贝数比例理论值与实测值
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