人体益生菌丁酸菌群检测方法的开发

人体益生菌丁酸菌群检测方法的开发摘要人体肠道微生物对于人体有着不可或缺的重要意义，而现今主要的检测手段有下一代测序技术(NGS)、传统培养法、荧光原位杂交(FISH)和实时荧光定量PC℉等，但这些研究方法有着操作繁琐、持续时间长、精度低、成本高、专业化程度高等缺点。我们本次研究准备开发一种TagMan gPCR方法，该方法有着较高的精确度和灵敏度，可以为丁酸产生菌群的研究提供更可靠的数据。我们本次研究检测的菌种是产丁酸菌群，为了设计出更准确，特异性更高的引物探针，通过NCBI的基因数据库得到产丁酸菌群的16 s rRNA,通过Integrated DNA Technology中的RT-qPCR引物探针设计工具设计出对应的上、下游引物和探针组合。由于需要保证检测结果的准确，因此样本为人体粪便样本，采用QIAamp PowerFecal Pro DNA的样本试剂盒。采用粪便微生物DNA提取试剂盒提取微生物基因组全DNA,先用准备好的引物进行普通PCR扩增检测，经琼脂糖凝胶电泳验证后，再采用qPCR检测验证是否符合实验要求。本实验通过设计出产丁酸菌群16SRNA的引物探针组合，检测时间快速，通常在2个小时内就可完成：而且其操作简单，不需要二代测序文库的复杂构建，仅仅需要可靠的测试人员和检测结果，适用于各个领域的广泛微生物检测。关键词：实时荧光定量PCR:产丁酸菌群：肠道微生物：引物探针AbstractHuman gut microorganisms are of indispensable importance to thehuman body,and the main detection methods today include next-generationsequencing technology (NGS),traditional culture methods,fluorescence insitu hybridization(FISH)and real-time PCR,but these research methods havethe disadvantages of cumbersome operation,long duration,low precision,high cost and high degree of specialization.In this study,we intend to developa TaqMan qPCR method with high accuracy and sensitivity,which canprovide more reliable data for the study of butyric acid producing flora.In order to design more accurate and more specific primer probes,the16s rRNA of butyric acid flora was obtained through the genetic database ofNCBI,and the corresponding upstream and downstream primer and probecombinations were designed through the RT-qPCR primer probe design toolin Integrated DNA Technology.Due to the need to ensure accurate test results,the sample was a human stool sample using the QIAamp PowerFecal ProDNA sample kit.The whole DNA of the microbial genome was extracted byfecal microbial DNA extraction kit,and the prepared primers were first usedfor ordinary PCR amplification detection,and after verification by agarose gelelectrophoresis,then qPCR detection was used to verify whether it met theexperimental requirements.In this experiment,the primer probe combination of butyric acid flora16S rRNA was designed,and the detection time was fast,usually completedwithin 2 hours.Moreover,it is simple to operate,does not require thecomplex construction of next-generation sequencing libraries,requiresreliable testers and test results,and is suitable for a wide range of microbialtests in various fields.Key words:Real-time PCR;Butyric acid-producing flora;gut microbes;Primer probes目录引言。第一章文献综述…。21.1立题背景21.2肠道菌群.21.3厚壁菌门简介.….31.4厚壁杆菌与人类健康的关系41.4.1肥胖与厚壁杆菌的关系44444444444444441.4.2腾食纤维的调节作用…1.4.3防治非酒精性脂肪肝NAFLD).51.5核酸检测技术1.5.1qPCR技术51.5.2RT-qPCR技术61.6研究意义…7第二章厚壁菌门实时荧光PCR检测方法.…2.1实验仪器设备及试剂..82.1.1仪器设备.82.1.2主要试剂.。82方法.2.2.1引物及探针的设计与合成82.2.2粪便DNA的提取.112.2.3反应体系优化..132.2.4琼脂凝胶电泳验证132.2.5PCR产物的回收142.2.6阳性质粒的制作…142.2.7RT-qPCR定量检测DNA.152.2.8RT-qPCR定量检测样本15第三章结果与分析173.1引物探针设计结果….173.2厚壁菌门16 s rRNA基因提取结果.183.3实时荧光定量PCR检测效果…183.4引物和探针检测效率和标准曲线制作193.5实时荧光定量PC检测样品中的厚壁菌门20第四章结论…22参考文献24致谢27引言“微生物群”的起源可以追溯到1900年代初。结果发现，包括细菌、酵母菌和病毒在内的大量微生物共存于人体的各个部位（肠道、皮肤、肺、口腔）[1]。此外，人类微生物群，也被称为“隐藏的器官”，贡献的遗传信息是整个人类基因组的150倍以上[2]。