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秦倩倩, 张玲, 王国庆. 实时荧光PCR定量检测粪便中Akkermansia muciniphila方法研究[J]. 四川大学学报(医学版), 2018, 49(1): 93-97.
引用本文: 秦倩倩, 张玲, 王国庆. 实时荧光PCR定量检测粪便中Akkermansia muciniphila方法研究[J]. 四川大学学报(医学版), 2018, 49(1): 93-97.
QIN Qian-qian, ZHANG Ling, WANG Guo-qing. Quantitative Detection of Akkermansia muciniphila in Fecal Samples by Real-time PCR[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(1): 93-97.
Citation: QIN Qian-qian, ZHANG Ling, WANG Guo-qing. Quantitative Detection of Akkermansia muciniphila in Fecal Samples by Real-time PCR[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(1): 93-97.

实时荧光PCR定量检测粪便中Akkermansia muciniphila方法研究

Quantitative Detection of Akkermansia muciniphila in Fecal Samples by Real-time PCR

  • 摘要: 目的 建立实时荧光PCR定量检测粪便中Akkermansia muciniphila (A. muciniphila)的方法,为A. muciniphila的定量检测及深入研究提供技术支持。 方法 建立SYBR Green Ⅰ 荧光PCR 定量检测A. muciniphila的方法,利用A. muciniphila标准品制备标准曲线,评价该方法的灵敏度、特异性、抗干扰性及重复性,并对30例人粪便样本进行检测。 结果 标准曲线线性关系良好(R2=0.999);方法灵敏度可达到1.0×102CFU/mL;该方法可特异性扩增A. muciniphila,不受其他肠道细菌的影响;组内和组间重复性较好,Ct值的变异系数均小于2%;30例人粪便样品中A. muciniphila阳性率为73%,Ct值为17.40~31.77。 结论 本研究建立的实时荧光PCR定量检测粪便中A. muciniphila的方法简便、快速、经济,且具有良好的灵敏度、特异性、抗干扰性和重复性,适用于实际粪便样品的检测。

     

    Abstract: Objective To develop a real-time fluorescence PCR assay for quantitative detection of Akkermansia muciniphila (A. muciniphila) in fecal samples, providing technical support for quantitative detection and deep research of A.muciniphila. Methods The quantitative detection method for A.muciniphila were established by using SYBR Green Ⅰ; The standard curve was made using A.muciniphila standards; The sensitivity, specificity, anti-interference and repeatability of this assay were analyzed; 30 human fecal samples were detected by the established method. Results The standard curve showed a good linear relationship with R2=0.999; The sensitivity of this assay reached to 1.0×102CFU/mL; This method could amplify A.muciniphila specifically, not affected by other intestinal bacteria; Repeatability of intra-group and inter-group were good with the coefficient of variation (CV)of Ct value lower than 2%; The positive rate of A.muciniphila in 30 human fecal samples was 73%, while its Ct value ranged from 17.40 to 31.77. Conclusion These results indicated that the established real-time PCR method for A. muciniphila’ quantitative detection was simple, rapid and economical, good in sensitivity, specificity, anti-interference, repeatability and suitable for the detection of A.muciniphila in faeces.

     

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