T-RFLP快速分析慢性腹泻患者肠道菌群及其应用研究

Rapid Analysis of Intestinal Microflora in Chronic Diarrhea Patients and Application Study by T-RFLP.

目的建立快速检测分析人体肠道菌群的方法,协助慢性腹泻病原学诊断。方法上游引物的5'端用6-FAM进行荧光标记,PCR 反应结束后选择适当的限制性内切酶酶切消化,在测序仪上通过毛细管电泳即可检测5'末端荧光标记的限制性片段。利用不同细菌产生的不同峰谱达到快速对细菌定性、定量的目的。对30例慢性腹泻患者和22例健康人进行肠道菌群分析,从而探讨菌群变化情况。结果该法能够对人体肠道菌群进行定性和定量分析,30例腹泻患者均发现肠道菌群紊乱。结论该法快速、特异、敏感。一个样品中能同时检测多种细菌,是一种很有希望的新技术。

Objective To establish a new method to rapidly detect and analyse the microfolra in the human intestinal tract and determine the etiological diagnosis of chronic diarrhea.Metheds The forward PCR primer was labeled with 6 - carboxyfluorescein amino bexy （6 - FAM） fluorescent dye to detect the terminal fragment of the PCR products after digestion by restricition enzymes. The restriction fragment were analysed by capillary electrophoresis using an antomated DNA sequencer. Different bacteria will be analysed qualitatively and quantitatively through their different patterns rapidly. The method was used for the detection of intestinal micmfloras of 30 cases with chronic diarrhea and 22 normal controls to explore the bacterial population dynamics of the human intestines. Results Predominant bacteria in human intestinal tract were successfully identiffed by this rapid molecular technique.Intestinal micmflora in all patients was markedly disturbed.Conclusion This protocol is rapid, specific, sensitive and capable of identifying multiple organisms in a single sample.This technology is of great utility for determining the clinical diagnosis and therapy.