【摘要】：金屬材料在腐蝕過程中受到微生物腐蝕的影響,硫酸鹽還原菌(sulfate-reducing bacteria,SRB)是一種能促進(jìn)金屬腐蝕的微生物。本文旨在通過以硫酸鹽還原菌厭氧培養(yǎng)體系的建立為基礎(chǔ),在成功培養(yǎng)SRB菌株的前提下,利用兩種核酸擴(kuò)增技術(shù)——聚合酶鏈?zhǔn)椒磻?yīng)(PCR)和重組酶聚合酶擴(kuò)增反應(yīng)(RPA)建立快速檢測SRB菌株的方法。通過以厭氧操作箱和厭氧培養(yǎng)盒為主的厭氧培養(yǎng)方法,培養(yǎng)了購自中國普通微生物菌種保藏管理中心的Desulfovibrio fructosivorans(Df,菌株編號(hào)3468)和獲贈(zèng)于中國科學(xué)院海洋研究所的Desulfovibrio vulgaris(Dv),Desulfovibrio caledoniensis(Dc)及7株采集分離后未鑒定的硫酸鹽還原菌。分別以硫酸鹽還原菌16s rDNA中的特異性保守片段和SRB特異性基因異化亞硫酸鹽還原酶(DSR)、腺苷酰硫酸還原酶(APS)為模板設(shè)計(jì)了11對(duì)引物。通過聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase Chain Reaction,PCR)以標(biāo)準(zhǔn)菌株Desulfovibrio caledoniensis(Dc)基因組DNA為模板驗(yàn)證引物的有效性并用多種SRB菌株以及大腸桿菌DH5α基因組DNA為模板驗(yàn)證引物的特異性,找到了2對(duì)能夠特異性擴(kuò)增部分SRB基因組DNA的引物。在PCR驗(yàn)證的2對(duì)特異性引物的基礎(chǔ)上,初步探索了利用重組酶聚合酶擴(kuò)增反應(yīng)(recombinase polymerase amplification,RPA)檢測SRB的方法,設(shè)計(jì)了4對(duì)RPA引物,經(jīng)過驗(yàn)證沒有獲得特異性檢測SRB的引物,但發(fā)現(xiàn)其中一對(duì)引物可用于PCR方法檢測SRB,為將來優(yōu)化RPA反應(yīng)體系及引物設(shè)計(jì)提供了重要參考。
[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 偽 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.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q93;Q78

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本文編號(hào)：2508197