双歧杆菌地高辛标记寡核苷酸基因探针制备和应用研究

目的探讨地高辛标记寡核苷酸基因探针应用于微生态研究的可行性和实用性。方法制备双歧杆菌属和部分种的地高辛标记16S rRNA寡核苷酸探针,初步应用于微生态制剂鉴定和临床肠道微生态检测,评价寡核苷酸探针杂交在肠道微生态研究和检测中的应用价值。结果地高辛标记寡核苷酸探针具有较好的特异性与灵敏度：地高辛标记的双歧杆菌属和种的共6种寡核苷酸基因探针与标准菌株杂交后灵敏度和特异度分别为属探针95％、75％,青春双歧87．5％、90％,两歧双歧87．5％、87．5％,短双歧87．5％、92．5％,婴儿双歧75％、95％,长双歧75％、100％。结论寡核苷酸基因探针用于肠道细菌的鉴定显示出一定前景,加大探针的种类与扩大调查范围有可能使该技术替代现有细菌培养技术。     

用电镜原位杂交技术对玉米中期染色体中RNA的研究

用生物素标记的玉米１８ＳｒＲＮＡ、小麦５ＳｒＲＮＡ 及ｔＲＮＡ 的ｃＤＮＡ 作为探针,在Ｋ４Ｍ 树脂包埋的玉米（Ｚｅａ ｍ ａｙｓ）根尖超薄切片上进行原位杂交。杂交后,用与１０ ｎｍ 金颗粒相连的亲和素对杂交子在电镜下进行检测。结果发现,在玉米中期染色体中存在有１８ＳｒＲＮＡ、５ＳｒＲＮＡ 和ｔＲＮＡ 分子,这些ＲＮＡ分子在染色体中的分布是随机的,即这些ＲＮＡ 分子在染色体的内部和周边均有分布。５ＳｒＲＮＡ 和ｔＲ－ＮＡ 在染色体中和细胞质中的含量基本相等,而１８ＳｒＲＮＡ 在染色体中的含量则明显高于细胞质。这一结果表明染色体中的ＲＮＡ 一部分来自于核仁,而另一部分则来自于核质     

ITS序列同源性在苏云金芽孢杆菌分型中的应用研究

ＰＣＲ扩增了苏云金芽孢杆菌９个亚种的１６Ｓ－２３Ｓ ｒＲＮＡ基因转录间隔（ＩＴＳ）片段,它们的长度均为１４４碱基;序列同源性分析结果批出这９个亚种及其它亚种的ＩＴＳ序列高度相似,说明１６Ｓ－２３Ｓ ｒＲＮＡ基因的ＩＴＳ序列不适于苏云金孢杆菌亚种的分型。     

ITS序列同源性在苏云金芽孢杆菌分型中的应用研究*

ＰＣＲ扩增了苏云金芽孢杆菌９个亚种的１６Ｓ－２３５ｒＲＮＡ基因转录间隔（ＩＴＳ）片段,它们的长度均为１４４碱基;序列同源性分析结果指出这９个亚种及其它亚种的ＩＴＳ序列高度相似,说明１６Ｓ－２３５ ｒＲＮＡ基因的ＩＴＳ序列不适于苏云金芽抱杆菌亚种的分型。     

用地高辛标记原位杂交技术定位黄鳝rRNA基因于二价染色体3q12—q24和7…

以地高辛标记重组质粒ＰＴＡ７１中所含的小麦ｒＲＮＡ基因作为探针,与ＥｃｏＲＩ酶切的黄鳝核基因组点ＤＮＡ经Ｓｏｕｔｈｅｒｎ杂交,呈现２条带,片段长度分别为１２．８ｋｂ和４．６ｋｂ。再运用染色体原位杂交技术及杂交一多重带显带技术,定位ｒＲＮＡ基因于黄鳝二价染色体３ｑ１２－ｑ２４和７ｑ１４－ｑ２６两个区间,其分布位点与硝酸银染法结果相符。此外,还讨论了在黄鳝二价体上开展基因定位研究的突出优点。     

用地高辛标记原位杂交技术定位黄鳝rRNA基因于二价染色体3q12─q24和7q14─q26

以地高辛标记重组质粒ＰＴＡ７１中所含的小麦ｒＲＮＡ基因作为探针,与ＥｃｏＲＩ酶切的黄鳝核基因组总ＤＮＡ经Ｓｏｕｔｈｅｒｎ杂交,呈现２条带,片段长度分别为１２．８ｋｂ和４．６ｋｂ。再运用染色体原位杂交技术及杂交后多重带显带技术,定位ｒＲＮＡ基因于黄鳝二价染色体３ｑ１２－ｑ２４和７ｑ１４－ｑ２６两个区间,其分布位点与硝酸银染色法结果相符。此外,还讨论了在黄鳝二价体上开展基因定位研究的突出优点。     

Frankia菌在非豆科放线结瘤植物根际内的存活与分布

提取Ｆｒａｎｋｉａ菌１６ＳｒＲＮＡ,制备具专一性的寡核苷酸探针,通过同源杂交,在人工接种的木麻黄根际内检测出痕量Ｆｒａｎｋｉｓ菌结果表明,Ｆｒａｎｋｉａ可在环境因子的作用下被动迁移不论接种点的位置如何,经６个月的时间稳定后,其分布状态基本相同,主要集中于土层下８～３０ｃｍ处     

青藏高原近缘野生大麦5S rRNA基因染色体原位杂交定位

采用原位杂交技术,以５Ｓ ｒＲＮＡ基因为探针,对产于青藏高原的４份近缘野生大麦和栽培大麦,即：二棱野生大麦Ｈｏｒｄｅｕｍ ｖｕｌｇａｒｅ Ｌ．ｓｓｐ．ｓｐｏｎｔａｎｅｕｍ（Ｋｏｃｈ）Ｈｓｕｅ,六棱野生大麦Ｈ．ｖｕｌｇａｒｅ Ｌ．ｓｓｐ．ａｇｒｉｏｃｒｉｔｈｏｎ（Ａｂｅｒｇ） Ｈｓｕｅ,六棱瓶形野生大麦Ｈ．ｖｕｌｇａｒｅ Ｌ．ｓｓｐ．ａｇｒｉｏｃｒｉｔｈｏｎ ｖａｒ．ｌａｇｕｎｃｕｌｉｆｏｒｍ（Ｂａｋｈｔ） Ｈｓｕｅ,栽培大麦Ｈ．ｖｕｌｇａｒｅ．Ｌ．进行了研究,将杂交结果进行观察与统计,并建立起５Ｓ ｒＲＮＡ基因定位的模式图。结果表明５Ｓ ｒＲＮＡ基因在染色体上的位点呈现动态变化,由二棱野生大麦、六棱瓶形野生大麦到六棱野生大麦、栽培大麦、位点数目有递增的趋势,而且位置也发生了某些改变。探讨了５Ｓ ｒＲＮＡ基因进化     

原位杂交及原位PCR检测幽门螺杆菌

本研究建立了检测幽门螺杆菌的原位杂交和原位ＰＣＲ技术,采用ＰＣＲ掺入的方法标记原位杂交的生物素探针,６份ＨＰ阳性胃活检组织冰冻切片杂交均为阳性,６份阴性对照组织为阴性。９／１２份ＨＰ阳性对照组织石蜡切片杂交阳性,２份阴性对照切片为阴性。３份阳性对照猫胃粘膜冰冻切片杂交也是阳性。原位ＰＣＲ的引物来自ＨＰ的１６ｓｒＲＮＡ基因,在扩增过程掺入生物素基因。４／４例ＨＰ阳性人胃粘膜冰冻切片原位扩增阳性,２／     

荧光原位杂交技术对土壤石油降解菌的检测

从辽河油田样品中筛选出一株高效石油降解菌,经鉴定为地衣芽孢杆菌。针对其16S rRNA设计寡核苷酸探针。荧光原位杂交(FISH)技术利用寡核苷酸探针检测特定细胞内的互补核苷酸序列。通过对纯菌和泥浆中地衣芽孢杆菌的FISH进行优化,得到泥浆中地衣芽孢杆菌的荧光原位杂交实验条件：样品固定时间17 h,杂交温度46 ℃,杂交时间3 h,杂交液中去离子甲酰胺浓度35%,冲洗缓冲液中与去离子甲酰胺对应的NaCl的浓度88 mmol·L^-1。运用上述FISH技术监测生物泥浆反应器中地衣芽孢杆菌量的变化,并与泥浆中含油率的变化进行比较,二者的变化情况符合微生物降解石油的趋势,为监测含油污泥中微生物的变化提供了一种可行的技术。     

Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora.

Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.     

Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora.

Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.     

Taxon-specific probes for the cellulolytic genus Fibrobacter reveal abundant and novel equine-associated populations.

A total of six 16S rRNA targeted oligonucleotide probes were used to quantify Fibrobacter abundance and diversity in the gastrointestinal contents of a pony. Approximately 12% of the total 16S rRNA extracted from cecal contents hybridized with a Fibrobacter genus-specific probe and a Fibrobacter succinogenes species-specific probe. However, no significant hybridization was observed with a probe for the species. Fibrobacter intestinalis or with three probes for F. succinogenes subspecies. This suggested the presence of a previously undescribed population of F. succinogenes-like organisms. Novel lineages of F. succinogenes were subsequently identified by using PCR primers specific for the genus to amplify sequences coding for 16S rRNA from DNA extracted from cecal contents. Sequences of the cloned amplification products were shown to be affiliated with F. succinogenes but represented two distinct, and novel, lines of descent within the species.     

Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.

Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.     

Development of multi-color FISH method for analysis of seven Bifidobacterium species in human feces

We have developed a multi-color fluorescence in situ hybridization (FISH) method which detects, by a single reaction, all seven species of Bifidobacterium (B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. dentium, and B. longum), the dominant bacteria in human feces. First, eight new types of oligonucleotide probe were designed, complementary with the 16S rRNA sequence specific to genus Bifidobacterium and each bifidobacterial species described above. Using whole cell hybridization, the fluorescent intensity was measured against the bacterial species targeted by each probe, to show that each probe is specific to the targeted bacteria and that the relative fluorescent intensity (RFI) as an indicator of probe accessibility is high at 61-117%. Then, bacterial species-specific probes were labeled with fluorochromes (FITC, TAMRA and Cy5) in seven different ways, singly or in combination. Using these probes, seven species of Bifidobacterium were differentially stained in mixed samples of cultured bacteria and feces from adult volunteers, proving the efficacy of this technique.     

Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences.

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.     

Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences.

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.     

Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.     

16S rRNA gene-based phylogenetic microarray for simultaneous identification of members of the genus Burkholderia

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia , an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia , Pandoraea , Ralstonia and Limnobacter . Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool PhyloDetect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmium-contaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.