针对浓香型白酒大曲样本,以16S r RNA基因为目的片段,分别采用16S r DNA克隆文库法和高通量测序法分析大曲中细菌微生物群落的组成,并通过丰度和多样性分析,比较了两种方法在研究大曲样品细菌多样性方面的适用性。结果表明,在门、纲、...针对浓香型白酒大曲样本,以16S r RNA基因为目的片段,分别采用16S r DNA克隆文库法和高通量测序法分析大曲中细菌微生物群落的组成,并通过丰度和多样性分析,比较了两种方法在研究大曲样品细菌多样性方面的适用性。结果表明,在门、纲、目、科和属的分类水平上,克隆文库方法检测大曲样本微生物得到4个门,4个纲,5个目,4个科,6个属;高通量测序得到13个门,22个纲,33个目,61个科,133个属。在门的水平上,克隆文库与高通量测序检测出优势类群的总数量与总丰度分别为3个(99.32%)和4个(98.61%),共有优势类群及其丰度分别为Firmicutes(88.88%和79.32%)、Proteobacteria(7.8%和15.04%)、Actinobacteria(2.72%和1.77%)。重复样本分析,得出的结果相似。克隆文库法与高通量测序法在反映样本微生物群落规模上差异较大,而在反应大曲样本中主要微生物的物种组成及数量比例上结果相近,特别是样本中优势微生物类群的结果基本相同。两种方法各具优势。展开更多
为有效防范惠州市水产养殖中细菌病害的发生,对惠州市水产养殖致病菌的耐药情况进行分析。首先,在28℃和37℃下,经6种抗生素平板筛选惠州市水产养殖采集水样中的耐药菌株;然后,随机选取分离得到的70株菌,对其16S rDNA进行测序,并获得46...为有效防范惠州市水产养殖中细菌病害的发生,对惠州市水产养殖致病菌的耐药情况进行分析。首先,在28℃和37℃下,经6种抗生素平板筛选惠州市水产养殖采集水样中的耐药菌株;然后,随机选取分离得到的70株菌,对其16S rDNA进行测序,并获得46株阳性扩增产物的测序结果;最后对16S r DNA测序结果进行分析,得出分离菌株中耐药菌的种属。研究发现,从养殖水样中分离得到的菌株分别属于产气单胞菌属、丛毛单胞菌属等8个属,养殖水体中的细菌主要对氨苄西林和诺氟沙星耐药,对四环素耐药率较低。因此,在水产养殖过程中,应尽量避免使用氨苄西林和诺氟沙星类抗生素,首选耐药率低的四环素作为水产养殖的药物。展开更多
The microbial community structures in an integrated two-phase anaerobic reactor（ITPAR）were investigated by 16 S r DNA clone library technology. The 75 L reactor was designed with a 25 L rotating acidogenic unit at t...The microbial community structures in an integrated two-phase anaerobic reactor（ITPAR）were investigated by 16 S r DNA clone library technology. The 75 L reactor was designed with a 25 L rotating acidogenic unit at the top and a 50 L conventional upflow methanogenic unit at the bottom, with a recirculation connected to the two units. The reactor had been operated for 21 stages to co-digest fruit/vegetable wastes and wheat straw, which showed a very good biogas production and decomposition of cellulosic materials. The results showed that many kinds of cellulose and glycan decomposition bacteria related with Bacteroidales,Clostridiales and Syntrophobacterales were dominated in the reactor, with more bacteria community diversities in the acidogenic unit. The methanogens were mostly related with Methanosaeta, Methanosarcina, Methanoculleus, Methanospirillum and Methanobacterium; the predominating genus Methanosaeta, accounting for 40.5%, 54.2%, 73.6% and 78.7% in four samples from top to bottom, indicated a major methanogenesis pathway by acetoclastic methanogenesis in the methanogenic unit. The beta diversity indexes illustrated a more similar distribution of bacterial communities than that of methanogens between acidogenic unit and methanogenic unit. The differentiation of methanogenic community composition in two phases, as well as pH values and volatile fatty acid（VFA） concentrations confirmed the phase separation of the ITPAR. Overall, the results of this study demonstrated that the special designing of ITPAR maintained a sufficient number of methanogens, more diverse communities and stronger syntrophic associations among microorganisms, which made two phase anaerobic digestion of cellulosic materials more efficient.展开更多
PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from...PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from different environments.(GTG)5 and BOXA1 R primer were selected for REP-PCR. Twenty arbitrary primers were used for RAPD to acquire DNA profiles from A. ferrooxidans. Both RAPD and REP-PCR produce complex banding patterns and show good discriminatory ability in differentiating closely related strains of A. ferrooxidans. The strains are clustered into 4 or 5 major groups and reveal genomic diversity using(GTG)5-PCR, BOX-PCR and RAPD analysis. Phylogenetic tree based on 16 S r DNA sequences of 23 strains and related strains shows that they are clustered into two distinct groups. Twelve strains are highly related to a new Acidithiobacillus named Acidithiobacillus ferrivorans. The results indicate that PCR-based methods are effective in revealing genetic diversity among A. ferrooxidans.展开更多
为了快速鉴别饲料中的狐狸、水貂、貉子和狗源性成分,根据线粒体16S r DNA种间保守序列,设计合成针对狐狸、水貂、貉子和狗的特异性引物和探针,通过对荧光PCR反应体系和反应条件的优化筛选,建立了多重实时荧光PCR方法,在同一PCR反应体...为了快速鉴别饲料中的狐狸、水貂、貉子和狗源性成分,根据线粒体16S r DNA种间保守序列,设计合成针对狐狸、水貂、貉子和狗的特异性引物和探针,通过对荧光PCR反应体系和反应条件的优化筛选,建立了多重实时荧光PCR方法,在同一PCR反应体系中可以同时完成4种动物源性成分检测。通过对15种其他物种的源性成分的检测,结果表明所设计的引物和探针具有很好的物种特异性,且灵敏度高,狐狸、水貂、貉子和狗的DNA检出限为0.01ng。对40份样品检测,其中5份检测出貉子、狐狸和水貂源性成分。结果表明,该方法可以有效地鉴别出饲料中狐狸、水貂、貉子和狗源性成分,同时适用于相关动物产品中。展开更多
本文采用16S r DNA高通量测序技术分析了不同温度发酵下虾油菌相的变化,采用酶谱电泳研究了虾油蛋白酶系的变化,并结合测定各质量指标以探索细菌在虾油发酵中的作用。研究发现虾油中细菌数量远高于真菌数量,显示了细菌对发酵的重要贡献...本文采用16S r DNA高通量测序技术分析了不同温度发酵下虾油菌相的变化,采用酶谱电泳研究了虾油蛋白酶系的变化,并结合测定各质量指标以探索细菌在虾油发酵中的作用。研究发现虾油中细菌数量远高于真菌数量,显示了细菌对发酵的重要贡献。虾油菌相复杂,且不同温度下菌相差异很大;发酵中菌相及蛋白酶系均呈现动态变化,25℃和35℃发酵的虾油中蛋白酶失活较少,且存在大量Tetragenococcus,Virgibacillus等产生良好风味成分的细菌;而45℃的虾油中蛋白酶失活严重,且检出的细菌与产生良好风味无关,感官评定也最差,因此,45℃不适合发酵虾油。发酵30 d时,虾油中的氨基酸态氮、无盐可溶性固形物、挥发性盐基氮(TVB-N)和三甲胺(TMA)等均达到峰值,此后各指标基本恒定,但菌相仍变化较大且有1种新蛋白酶出现,而且虾油风味继续改善。展开更多
为建立用于二代测序技术的最适细菌DNA提取方法。分别用磁珠法、热裂解法、试剂盒法(有溶菌酶处理和无溶菌酶处理)、超声波法、超声+热裂解法共6种方法提取等量混合的细菌DNA,特异性扩增其16S r RNA的V3-V4高变区基因,经二代测序并分析...为建立用于二代测序技术的最适细菌DNA提取方法。分别用磁珠法、热裂解法、试剂盒法(有溶菌酶处理和无溶菌酶处理)、超声波法、超声+热裂解法共6种方法提取等量混合的细菌DNA,特异性扩增其16S r RNA的V3-V4高变区基因,经二代测序并分析结果中的细菌种类及其相对含量分布。6种方法的测序结果中,细菌种类鉴定结果无明显差异;而在磁珠法和试剂盒法(有溶菌酶处理)结果中,不同细菌的含量分布最为接近样品中的真实情况。在6种方法中,磁珠法和试剂盒法(有溶菌酶处理)适合于二代测序技术中混合细菌DNA的提取。展开更多
从沈阳市7个区25份朝鲜族家庭制作的传统发酵辣白菜中分离出81株乳酸菌疑似菌株,初步鉴定34株为杆菌,47株为球菌。进一步采用16S r DNA序列分析对81株菌进行分子鉴定,通过序列分析进行属种鉴定。结果表明:81株菌均为乳酸菌,分别来自2个...从沈阳市7个区25份朝鲜族家庭制作的传统发酵辣白菜中分离出81株乳酸菌疑似菌株,初步鉴定34株为杆菌,47株为球菌。进一步采用16S r DNA序列分析对81株菌进行分子鉴定,通过序列分析进行属种鉴定。结果表明:81株菌均为乳酸菌,分别来自2个属6个种,45株为屎肠球菌,25株为植物乳杆菌,4株为干酪乳杆菌,3株为戊糖乳杆菌,2株为短乳杆菌,2株为坚强肠球菌。研究结果为我国东北辣白菜中乳酸菌作进一步研究奠定了基础。展开更多
为探究准好氧生物矿化垃圾床处理渗滤液过程中的微生物作用机理,研究建立了16S r DNA克隆文库及PCRRFLP技术以研究矿化垃圾反应器的细菌多样性。结果表明:矿化垃圾反应器细菌具有高度多样性,反应器内有36种细菌分别属于13个纲,变形菌纲...为探究准好氧生物矿化垃圾床处理渗滤液过程中的微生物作用机理,研究建立了16S r DNA克隆文库及PCRRFLP技术以研究矿化垃圾反应器的细菌多样性。结果表明:矿化垃圾反应器细菌具有高度多样性,反应器内有36种细菌分别属于13个纲,变形菌纲占绝对优势占70%(其中β-变形占56.5%,γ-变形菌纲占10%),拟杆菌纲、鞘氨醇菌纲,芽孢杆菌纲也具有一定优势;3%的Nitrosomonas属是渗滤液氨氮转化成亚硝酸盐氮的主要功能微生物,由于亚硝酸盐氧化菌不存在或丰度极低,因此造成反应器内亚硝酸盐积累;Thauera属(17%)和Thiobacillus denitrificans属(10%)是反应器内主要优势微生物属,是反应器内反硝化脱氮的功能微生物,由于Thauera属在好氧条件下具有反硝化特性,Thiobacillus denitrificans为严格自养反硝化菌,因此反应器脱氮主要途径为好氧反硝化、自养反硝化。展开更多
文摘针对浓香型白酒大曲样本,以16S r RNA基因为目的片段,分别采用16S r DNA克隆文库法和高通量测序法分析大曲中细菌微生物群落的组成,并通过丰度和多样性分析,比较了两种方法在研究大曲样品细菌多样性方面的适用性。结果表明,在门、纲、目、科和属的分类水平上,克隆文库方法检测大曲样本微生物得到4个门,4个纲,5个目,4个科,6个属;高通量测序得到13个门,22个纲,33个目,61个科,133个属。在门的水平上,克隆文库与高通量测序检测出优势类群的总数量与总丰度分别为3个(99.32%)和4个(98.61%),共有优势类群及其丰度分别为Firmicutes(88.88%和79.32%)、Proteobacteria(7.8%和15.04%)、Actinobacteria(2.72%和1.77%)。重复样本分析,得出的结果相似。克隆文库法与高通量测序法在反映样本微生物群落规模上差异较大,而在反应大曲样本中主要微生物的物种组成及数量比例上结果相近,特别是样本中优势微生物类群的结果基本相同。两种方法各具优势。
文摘为有效防范惠州市水产养殖中细菌病害的发生,对惠州市水产养殖致病菌的耐药情况进行分析。首先,在28℃和37℃下,经6种抗生素平板筛选惠州市水产养殖采集水样中的耐药菌株;然后,随机选取分离得到的70株菌,对其16S rDNA进行测序,并获得46株阳性扩增产物的测序结果;最后对16S r DNA测序结果进行分析,得出分离菌株中耐药菌的种属。研究发现,从养殖水样中分离得到的菌株分别属于产气单胞菌属、丛毛单胞菌属等8个属,养殖水体中的细菌主要对氨苄西林和诺氟沙星耐药,对四环素耐药率较低。因此,在水产养殖过程中,应尽量避免使用氨苄西林和诺氟沙星类抗生素,首选耐药率低的四环素作为水产养殖的药物。
基金supported by the Major Science and Technology Programs for Water Pollution Control and Management of China(No.2012ZX07205-001)the National Science and Technology Support Program(No.2008BADC4B18)
文摘The microbial community structures in an integrated two-phase anaerobic reactor（ITPAR）were investigated by 16 S r DNA clone library technology. The 75 L reactor was designed with a 25 L rotating acidogenic unit at the top and a 50 L conventional upflow methanogenic unit at the bottom, with a recirculation connected to the two units. The reactor had been operated for 21 stages to co-digest fruit/vegetable wastes and wheat straw, which showed a very good biogas production and decomposition of cellulosic materials. The results showed that many kinds of cellulose and glycan decomposition bacteria related with Bacteroidales,Clostridiales and Syntrophobacterales were dominated in the reactor, with more bacteria community diversities in the acidogenic unit. The methanogens were mostly related with Methanosaeta, Methanosarcina, Methanoculleus, Methanospirillum and Methanobacterium; the predominating genus Methanosaeta, accounting for 40.5%, 54.2%, 73.6% and 78.7% in four samples from top to bottom, indicated a major methanogenesis pathway by acetoclastic methanogenesis in the methanogenic unit. The beta diversity indexes illustrated a more similar distribution of bacterial communities than that of methanogens between acidogenic unit and methanogenic unit. The differentiation of methanogenic community composition in two phases, as well as pH values and volatile fatty acid（VFA） concentrations confirmed the phase separation of the ITPAR. Overall, the results of this study demonstrated that the special designing of ITPAR maintained a sufficient number of methanogens, more diverse communities and stronger syntrophic associations among microorganisms, which made two phase anaerobic digestion of cellulosic materials more efficient.
基金Project(2010CB630901)supported by the National Basic Research Program of China
文摘PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from different environments.(GTG)5 and BOXA1 R primer were selected for REP-PCR. Twenty arbitrary primers were used for RAPD to acquire DNA profiles from A. ferrooxidans. Both RAPD and REP-PCR produce complex banding patterns and show good discriminatory ability in differentiating closely related strains of A. ferrooxidans. The strains are clustered into 4 or 5 major groups and reveal genomic diversity using(GTG)5-PCR, BOX-PCR and RAPD analysis. Phylogenetic tree based on 16 S r DNA sequences of 23 strains and related strains shows that they are clustered into two distinct groups. Twelve strains are highly related to a new Acidithiobacillus named Acidithiobacillus ferrivorans. The results indicate that PCR-based methods are effective in revealing genetic diversity among A. ferrooxidans.
文摘为了快速鉴别饲料中的狐狸、水貂、貉子和狗源性成分,根据线粒体16S r DNA种间保守序列,设计合成针对狐狸、水貂、貉子和狗的特异性引物和探针,通过对荧光PCR反应体系和反应条件的优化筛选,建立了多重实时荧光PCR方法,在同一PCR反应体系中可以同时完成4种动物源性成分检测。通过对15种其他物种的源性成分的检测,结果表明所设计的引物和探针具有很好的物种特异性,且灵敏度高,狐狸、水貂、貉子和狗的DNA检出限为0.01ng。对40份样品检测,其中5份检测出貉子、狐狸和水貂源性成分。结果表明,该方法可以有效地鉴别出饲料中狐狸、水貂、貉子和狗源性成分,同时适用于相关动物产品中。
文摘本文采用16S r DNA高通量测序技术分析了不同温度发酵下虾油菌相的变化,采用酶谱电泳研究了虾油蛋白酶系的变化,并结合测定各质量指标以探索细菌在虾油发酵中的作用。研究发现虾油中细菌数量远高于真菌数量,显示了细菌对发酵的重要贡献。虾油菌相复杂,且不同温度下菌相差异很大;发酵中菌相及蛋白酶系均呈现动态变化,25℃和35℃发酵的虾油中蛋白酶失活较少,且存在大量Tetragenococcus,Virgibacillus等产生良好风味成分的细菌;而45℃的虾油中蛋白酶失活严重,且检出的细菌与产生良好风味无关,感官评定也最差,因此,45℃不适合发酵虾油。发酵30 d时,虾油中的氨基酸态氮、无盐可溶性固形物、挥发性盐基氮(TVB-N)和三甲胺(TMA)等均达到峰值,此后各指标基本恒定,但菌相仍变化较大且有1种新蛋白酶出现,而且虾油风味继续改善。
文摘为建立用于二代测序技术的最适细菌DNA提取方法。分别用磁珠法、热裂解法、试剂盒法(有溶菌酶处理和无溶菌酶处理)、超声波法、超声+热裂解法共6种方法提取等量混合的细菌DNA,特异性扩增其16S r RNA的V3-V4高变区基因,经二代测序并分析结果中的细菌种类及其相对含量分布。6种方法的测序结果中,细菌种类鉴定结果无明显差异;而在磁珠法和试剂盒法(有溶菌酶处理)结果中,不同细菌的含量分布最为接近样品中的真实情况。在6种方法中,磁珠法和试剂盒法(有溶菌酶处理)适合于二代测序技术中混合细菌DNA的提取。
文摘从沈阳市7个区25份朝鲜族家庭制作的传统发酵辣白菜中分离出81株乳酸菌疑似菌株,初步鉴定34株为杆菌,47株为球菌。进一步采用16S r DNA序列分析对81株菌进行分子鉴定,通过序列分析进行属种鉴定。结果表明:81株菌均为乳酸菌,分别来自2个属6个种,45株为屎肠球菌,25株为植物乳杆菌,4株为干酪乳杆菌,3株为戊糖乳杆菌,2株为短乳杆菌,2株为坚强肠球菌。研究结果为我国东北辣白菜中乳酸菌作进一步研究奠定了基础。
文摘为探究准好氧生物矿化垃圾床处理渗滤液过程中的微生物作用机理,研究建立了16S r DNA克隆文库及PCRRFLP技术以研究矿化垃圾反应器的细菌多样性。结果表明:矿化垃圾反应器细菌具有高度多样性,反应器内有36种细菌分别属于13个纲,变形菌纲占绝对优势占70%(其中β-变形占56.5%,γ-变形菌纲占10%),拟杆菌纲、鞘氨醇菌纲,芽孢杆菌纲也具有一定优势;3%的Nitrosomonas属是渗滤液氨氮转化成亚硝酸盐氮的主要功能微生物,由于亚硝酸盐氧化菌不存在或丰度极低,因此造成反应器内亚硝酸盐积累;Thauera属(17%)和Thiobacillus denitrificans属(10%)是反应器内主要优势微生物属,是反应器内反硝化脱氮的功能微生物,由于Thauera属在好氧条件下具有反硝化特性,Thiobacillus denitrificans为严格自养反硝化菌,因此反应器脱氮主要途径为好氧反硝化、自养反硝化。
