用细菌16S rRNA荧光定量PCR法检测肠道菌群的变化

Detecting the variation intestinal microflora by 16S rRNA fluorescent quantificative PCR

目的应用细菌的16S rRNA序列设计双歧杆菌、大肠埃希菌及乳酸杆菌的引物并对肠道的3种细菌进行定量测定。方法收集轮状病毒肠炎患儿及正常对照组的粪便标本提取DNA。取准确定量的3种细菌经系列稀释后抽提细菌的DNA做荧光定量PCR,制作出标准曲线,待测样品同时进行PCR反应并和标准曲线进行比较,获得各样品中3种细菌的量。结果患儿肠道中双歧杆菌和乳酸杆菌的数量较正常儿童明显减低,而大肠埃希菌的数量差异无显著性。与其他文献报道的用细菌培养的方法所得结果一致。结论荧光定量PCR是一种特异性高、敏感性强的定量方法。可正确定量肠道中的细菌数量。

Objective To design the primes of Bifidobacteria, Escherichia coli and Lactobacillus by 16S rRNA sequence of bacteria and detect three bacteria in the intestine by fluorescent quantificative PCR. Methods To collect the feces of the children with rotavirus enteritis and the control and to extract the DNA. The standard curve of fluorescent quantificative PCR was developed by using a series of dilution of bacteria DNA that extracted from the three accurate bacteria. The unknown samples were measured by PCR and compared with the standard curve. Results To comparison with the healthy children,the number of Bifidobacteria and Lactobacillus appeared to be declined in patients（P〈0.05）. However the number of Escherichia coli had no significant variation（P〉0.05） ,and the range of bacteria by fluorescent quantificative PCR was nearly equal with the result by the cultivation techniques from oth- er document. Conclntion Fluorescent quantificative PCR was a method with high specificity and sensitivity ,it could be used for the quantification of bacteria in the intestine.