基于核酸扩增的硫酸盐还原菌分子诊断研究
发布时间：2019-06-30 19:47
【摘要】：金属材料在腐蚀过程中受到微生物腐蚀的影响,硫酸盐还原菌(sulfate-reducing bacteria,SRB)是一种能促进金属腐蚀的微生物。本文旨在通过以硫酸盐还原菌厌氧培养体系的建立为基础,在成功培养SRB菌株的前提下,利用两种核酸扩增技术——聚合酶链式反应(PCR)和重组酶聚合酶扩增反应(RPA)建立快速检测SRB菌株的方法。通过以厌氧操作箱和厌氧培养盒为主的厌氧培养方法,培养了购自中国普通微生物菌种保藏管理中心的Desulfovibrio fructosivorans(Df,菌株编号3468)和获赠于中国科学院海洋研究所的Desulfovibrio vulgaris(Dv),Desulfovibrio caledoniensis(Dc)及7株采集分离后未鉴定的硫酸盐还原菌。分别以硫酸盐还原菌16s rDNA中的特异性保守片段和SRB特异性基因异化亚硫酸盐还原酶(DSR)、腺苷酰硫酸还原酶(APS)为模板设计了11对引物。通过聚合酶链式反应(Polymerase Chain Reaction,PCR)以标准菌株Desulfovibrio caledoniensis(Dc)基因组DNA为模板验证引物的有效性并用多种SRB菌株以及大肠杆菌DH5α基因组DNA为模板验证引物的特异性,找到了2对能够特异性扩增部分SRB基因组DNA的引物。在PCR验证的2对特异性引物的基础上,初步探索了利用重组酶聚合酶扩增反应(recombinase polymerase amplification,RPA)检测SRB的方法,设计了4对RPA引物,经过验证没有获得特异性检测SRB的引物,但发现其中一对引物可用于PCR方法检测SRB,为将来优化RPA反应体系及引物设计提供了重要参考。
[Abstract]:Metal materials are affected by microbial corrosion in the corrosion process. Sulfate reducing bacteria (sulfate-reducing bacteria,SRB) is a kind of microorganism which can promote metal corrosion. The purpose of this paper was to establish a rapid method for the detection of SRB strains by using two nucleic acid amplification techniques, polymerase chain reaction (PCR) and recombinant enzyme polymerase amplification (RPA), based on the establishment of anaerobic culture system of sulfate-reducing bacteria. Desulfovibrio fructosivorans (Df, strain No. 3468, purchased from the Storage Management Center of Common microbial bacteria in China, and 7 unidentified sulfate-reducing bacteria, which were collected and isolated from the Institute of Oceanography, Chinese Academy of Sciences, were cultured by means of anaerobics culture method, which was mainly composed of anaerobically operated box and anaerobically culture box (Desulfovibrio vulgaris (Dv), Desulfovibrio caledoniensis (Dc) strain No. 3468) and 7 strains collected and isolated from the Institute of Oceanography, Chinese Academy of Sciences. Eleven pairs of primers were designed using the specific conserved fragment of sulfate reducing bacteria 16s rDNA and the SRB specific gene alienation sulfite reducase (DSR), adenosine sulfate reductase (APS) as templates. Polymerase chain reaction (Polymerase Chain Reaction,PCR) using standard strain Desulfovibrio caledoniensis (Dc) genomic DNA as template to verify the effectiveness of primers and using various SRB strains and E. coli DH5 伪 genomic DNA as templates to verify the specificity of primers, two pairs of primers which could specifically amplify part of SRB genomic DNA were found. On the basis of two pairs of specific primers verified by PCR, the method of detecting SRB by recombinant enzyme polymerase (recombinase polymerase amplification,RPA (recombinase polymerase amplification,RPA) was preliminarily explored. Four pairs of RPA primers were designed. It was verified that there were no primers for specific detection of SRB, but it was found that one pair of primers could be used to detect SRB, by PCR method, which provided an important reference for optimizing RPA reaction system and primer design in the future.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q93;Q78
本文编号：2508197
[Abstract]:Metal materials are affected by microbial corrosion in the corrosion process. Sulfate reducing bacteria (sulfate-reducing bacteria,SRB) is a kind of microorganism which can promote metal corrosion. The purpose of this paper was to establish a rapid method for the detection of SRB strains by using two nucleic acid amplification techniques, polymerase chain reaction (PCR) and recombinant enzyme polymerase amplification (RPA), based on the establishment of anaerobic culture system of sulfate-reducing bacteria. Desulfovibrio fructosivorans (Df, strain No. 3468, purchased from the Storage Management Center of Common microbial bacteria in China, and 7 unidentified sulfate-reducing bacteria, which were collected and isolated from the Institute of Oceanography, Chinese Academy of Sciences, were cultured by means of anaerobics culture method, which was mainly composed of anaerobically operated box and anaerobically culture box (Desulfovibrio vulgaris (Dv), Desulfovibrio caledoniensis (Dc) strain No. 3468) and 7 strains collected and isolated from the Institute of Oceanography, Chinese Academy of Sciences. Eleven pairs of primers were designed using the specific conserved fragment of sulfate reducing bacteria 16s rDNA and the SRB specific gene alienation sulfite reducase (DSR), adenosine sulfate reductase (APS) as templates. Polymerase chain reaction (Polymerase Chain Reaction,PCR) using standard strain Desulfovibrio caledoniensis (Dc) genomic DNA as template to verify the effectiveness of primers and using various SRB strains and E. coli DH5 伪 genomic DNA as templates to verify the specificity of primers, two pairs of primers which could specifically amplify part of SRB genomic DNA were found. On the basis of two pairs of specific primers verified by PCR, the method of detecting SRB by recombinant enzyme polymerase (recombinase polymerase amplification,RPA (recombinase polymerase amplification,RPA) was preliminarily explored. Four pairs of RPA primers were designed. It was verified that there were no primers for specific detection of SRB, but it was found that one pair of primers could be used to detect SRB, by PCR method, which provided an important reference for optimizing RPA reaction system and primer design in the future.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q93;Q78
【参考文献】
相关期刊论文 前2条
1 吴龙益;潘翠;周漪;;微生物对装备的影响[J];装备环境工程;2006年05期
2 刘光洲,吴建华;海洋微生物腐蚀的研究进展[J];腐蚀与防护;2001年10期
，本文编号：2508197
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