Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise co...Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as ＂bait＂： Fusobacterium nucleatum （F. nucleatum） whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans （S. mutans） whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the ＂bait＂ oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The ＂prey＂ bacterial mixtures （including known species or natural saliva samples） were added, unbound ceils were washed off after the incubation period and the remaining cells were eluted using 0.2 mol.L1 glycine. Genomic DNA was extraeted, subjeeted to 16S rRNA PCR amplification and separation of the resulting PCR produets by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F.. nucleatum adhered to a variety of bacterial species including uncultivable and uneharacterized onesl This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.展开更多
采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术对盐质量浓度5 g/100 m L和19 g/100 m L腌制麻竹笋的微生物区系进行研究。结果表明,经DNA提取、巢式PCR、DGG...采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术对盐质量浓度5 g/100 m L和19 g/100 m L腌制麻竹笋的微生物区系进行研究。结果表明,经DNA提取、巢式PCR、DGGE电泳和克隆测序后,从低盐质量浓度(5 g/100 m L)腌制笋中分离出4条明显的亮带,经鉴定分别为食窦魏斯氏菌(Weissella cibaria)、乳球菌属(Lactococcus sp.)、魏斯氏菌属(Weissella sp.)和乳酸乳球菌(Lactococcus lactis);从高盐质量浓度(19 g/100 m L)腌制笋中分离出5条明显的亮带,经鉴定分别为绿色气球菌(Aerococcus viridans)、赖氨酸芽孢杆菌属(Lysinibacillus sp.)、未得到培养的细菌(uncultured bacterium)、厌氧芽孢杆菌属(Anoxybacillus sp.)和芽孢杆菌属(Bacillus sp.);低盐腌制笋的优势菌多为益生菌,而高盐腌制笋的优势菌则多为抗性较强的菌。基于16S r DNA的PCR-DGGE技术为分析腌制麻竹笋中微生物多样性提供了一条可靠、快速的有效途径。展开更多
为了获得完整、高纯度的竹林土壤微生物总DNA,采用改良的十二烷基硫酸钠-高盐抽提法对5种竹林土壤进行DNA提取,通过DNA凝胶回收试剂盒纯化粗提的DNA,利用细菌16 S rDNA基因的V3区引物对纯化的DNA进行了聚合酶链式反应-变性梯度凝胶电泳(...为了获得完整、高纯度的竹林土壤微生物总DNA,采用改良的十二烷基硫酸钠-高盐抽提法对5种竹林土壤进行DNA提取,通过DNA凝胶回收试剂盒纯化粗提的DNA,利用细菌16 S rDNA基因的V3区引物对纯化的DNA进行了聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)验证。结果表明,该方法所需土壤样品少,抽提的DNA片段均在23kb以上,完整性好;采用DNA凝胶回收试剂盒纯化能够有效去除粗提DNA中大部分杂质,获得高质量的DNA;以此DNA为模板进行DGGE检测所反映的微生物信息量丰富。展开更多
基金supported by Chinese State Scholarship Fund to R. WangUS National Institutes of Health (NIH) Grants DE020102 and GM95373 to W. Shi
文摘Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as ＂bait＂： Fusobacterium nucleatum （F. nucleatum） whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans （S. mutans） whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the ＂bait＂ oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The ＂prey＂ bacterial mixtures （including known species or natural saliva samples） were added, unbound ceils were washed off after the incubation period and the remaining cells were eluted using 0.2 mol.L1 glycine. Genomic DNA was extraeted, subjeeted to 16S rRNA PCR amplification and separation of the resulting PCR produets by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F.. nucleatum adhered to a variety of bacterial species including uncultivable and uneharacterized onesl This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.
文摘采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术对盐质量浓度5 g/100 m L和19 g/100 m L腌制麻竹笋的微生物区系进行研究。结果表明,经DNA提取、巢式PCR、DGGE电泳和克隆测序后,从低盐质量浓度(5 g/100 m L)腌制笋中分离出4条明显的亮带,经鉴定分别为食窦魏斯氏菌(Weissella cibaria)、乳球菌属(Lactococcus sp.)、魏斯氏菌属(Weissella sp.)和乳酸乳球菌(Lactococcus lactis);从高盐质量浓度(19 g/100 m L)腌制笋中分离出5条明显的亮带,经鉴定分别为绿色气球菌(Aerococcus viridans)、赖氨酸芽孢杆菌属(Lysinibacillus sp.)、未得到培养的细菌(uncultured bacterium)、厌氧芽孢杆菌属(Anoxybacillus sp.)和芽孢杆菌属(Bacillus sp.);低盐腌制笋的优势菌多为益生菌,而高盐腌制笋的优势菌则多为抗性较强的菌。基于16S r DNA的PCR-DGGE技术为分析腌制麻竹笋中微生物多样性提供了一条可靠、快速的有效途径。
文摘为了获得完整、高纯度的竹林土壤微生物总DNA,采用改良的十二烷基硫酸钠-高盐抽提法对5种竹林土壤进行DNA提取,通过DNA凝胶回收试剂盒纯化粗提的DNA,利用细菌16 S rDNA基因的V3区引物对纯化的DNA进行了聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)验证。结果表明,该方法所需土壤样品少,抽提的DNA片段均在23kb以上,完整性好;采用DNA凝胶回收试剂盒纯化能够有效去除粗提DNA中大部分杂质,获得高质量的DNA;以此DNA为模板进行DGGE检测所反映的微生物信息量丰富。
