应用real-timePCR法快速定量人类粪便中双歧杆菌的研究

目的建立快速、准确从粪便标本中定量双歧杆菌的RT—PCR技术。方法传统培养定量法，普通PCR定量法，real—timePCR比较测量。结果（I）粪便标本前处理采取简单的离心和清洗、稀释步骤能去除粪便标本中的抑制物，实现不提取DNA直接进行PCR、real—time定量粪便中双歧杆菌。（2）本实验建立的PCR方法直接半定量粪便双歧杆菌技术在双歧杆菌值介于10^3～10^7CFU／ml时具有较好的分辨率，粪便标本普通PCR得理论菌数与培养得菌数值之间差异无显著性（P〉0．05）；real-timePCR直接定量双歧杆菌技术在双歧杆菌值介于10^1-10^7CFU／ml时具有较好的分辨率，粪便标本RT—PCR得理论菌数与培养得菌数值之间差异无显著性（P〉0．05）。结论利用PCR、real—timePCR直接半定量和定量粪便中的双歧杆菌可行。

A fast quantification for Bifidobacteria in human feces by real-time PCR

Objective To build a fast and accurate method of quantification for Bifidobacteria in human feces. Method Traditional culture, common PCR, real-time PCR were used. Result (1) The inhibitors in the feces samples were removed when the samples were centrifuged, washed and diluted,so that the Bifidobacteria in the feces samples could be directly quantitated by PCR and real-time PCR. (2) The direct semiquantitation by PCR established in this study showed a satisfactory distinguishability when Bifidobacteria in feces samples was between 10~3 ～10~7CFU /ml. There was no obvious difference between the result from RT-PCR quantification and that from cultured method (P > 0.05); The direct quantita-tion by PCR showed a satisfactory distinguishability when Bifidobacteria in feces samples was between 10~1 ～ 10~7CFU /ml, without obvious difference between the result from RT-PCR and that from cultured method (P > 0. 05). Conclusion Bifidobacteria in feces samples can be directly quantitated or semiquantitated by real-time PCR method or common PCR method.