快速评价肠道微生物的qPCR方法的建立  

A qPCR method for rapid detection of gut microorganisms

作　　者：梁海慧 张扬[2] 边爱淑 周凡入 李学龙 张秀峰 尹秀山 LIANG Haihui;ZHANG Yang;BIAN Aishu;ZHOU Fanru;LI Xuelong;ZHANG Xiufeng;YIN Xiushan(College of Pharmaceutical and Biological Engineering,Shenyang University of Chemical Technology,Shenyang 110142,Liaoning,China;Department of Gastroenterology,242 Affiliated Hospital of Shenyang Medical College,Shenyang 110034,Liaoning,China;IDM Biotechnology(Shenyang)Limited Company,Shenyang 110044,Liaoning,China)

机构地区：[1]沈阳化工大学制药与生物工程学院,辽宁沈阳110142 [2]沈阳医学院附属二四二医院消化内科,辽宁沈阳110034 [3]鉴微(沈阳)生物科技有限公司,辽宁沈阳110044

出　　处：《微生物学通报》2023年第11期5045-5057,共13页Microbiology China

基　　金：辽宁省振兴人才计划(XLYC2002027)。

摘　　要：【背景】近些年,16S rRNA基因测序与宏基因组分析常用于肠道微生物病原体检测。【目的】为了使检测不受限于高成本与耗时长的问题,基于荧光探针的实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR),建立一种评估人类肠道微生物群组成的平台用于检测肠道微生物丰度。【方法】从公共数据库筛选10种肠道中普遍存在的微生物分类群,使用20个粪便样本验证为10种靶标所设计的特异性引物与探针,最后通过比较qPCR方法和16S rRNA基因测序技术的检测结果来评估该平台的有效性。【结果】10对引物及其探针对靶标分类群具有特异性并且在HITdb数据库中靶向菌种的覆盖率超过70%;样本检测结果的变异系数(coefficient of variation,CV)小于10%,证明了该方法具有很高的稳定性;qPCR方法检测样本中物种的相对丰度与16S rRNA基因序列生物信息学分析结果大部分具有显著相关性(P<0.05)。【结论】本研究根据HITdb数据库设计的靶向微生物群的引物和探针检测到的粪便样本中微生物的相对丰度结果与16S rRNA基因测序结果相吻合,可以提供精确且低成本的肠道微生物群分析基础,同时也为临床诊断制定治疗方案提供了快速有效的参考信息。[Background]In recent years,16S rRNA gene sequencing and metagenomic analysis have been commonly used for detecting microbial pathogens in the gut.[Objective]To overcome the limitations of high cost and long detection time,we established a method for analyzing the composition of human gut microbiota based on real-time fluorescence quantitative PCR(qPCR),which can measure the abundance of gut microorganisms.[Methods]Ten microbial taxa commonly detected in the intestine were selected from public databases.We designed specific primers and probes for the ten targets and validated them using 20 fecal samples.Furthermore,we compared the results of qPCR and 16S rRNA gene sequencing to evaluate the performance of the established method.[Results]The ten pairs of primers and probes were specific to the targeted taxa,and the coverage of targeted bacteria in the HITdb database exceeded 70%.The coefficient of variation(CV)of sample detection results was less than 10%,indicating that the method was highly stable.The results of qPCR and 16S rRNA gene sequencing for measuring the microbial abundance were correlated(P<0.05).[Conclusion]The results of the relative abundance of microorganisms detected in fecal samples using the primers and probes designed based on the HITdb database were consistent with those obtained by 16S rRNA gene sequencing.This study provides an accurate and cost-effective method for analyzing gut microbiota and the reference information for clinical diagnosis and treatment.

关 键 词：肠道微生物 16S rRNA qPCR 菌群丰度 

分 类 号：R446.5[医药卫生—诊断学]