不同分子探针对赤潮样品检测效果的比较研究

采用荧光标记的DNA和lecfn探针对来自赤潮现场样品中的目标赤潮原因种进行了检测.结果表明这些探针能从比较复杂的现场样品背景中把目标赤潮生物识别出来.从检测效果上看,各种DNA探针对塔玛亚历山大藻、微小原甲藻和T.pulchellum标记识别的荧光信号强度相对于lectin探针要弱.Lectin探针与明视野下的显微镜计数结果呈现出很好的相关性,但lectin探针对目标藻的绝对栓出数量比明视野下的计数结果要低.lectin探针(ConA)和DNA探针(PM18S02、PM28S02)对现场样品中微小原甲藻的检测计数结果也有较好的相关性.表明在这些检测条件下,不同的探针对特定目标藻的检测结果具有可比性、相关性和可操作性.     

用细胞周期分析结合免疫印迹方法检测微型浮游植物细胞增长率的研究

采用基于流式细胞仪的细胞周期分析和免疫印迹方法来研究浮游植物的细胞增长率.检测和分析同步化后的有害浮游植物Takayama pulchellum,可知T.pulchellum的S期大约出现在10h左右.免疫印迹检测的结果显示抗微小原甲藻（Prorocentrum minimum）和抗Pfiesteria piscicida的PCNA抗体与T.pulchellum无明显的阳性杂交信号出现,表明这2种PCNA抗体不能作为检测T.pulchellum PCNA表达的抗体.Rubisco抗体免疫印迹反应的信号强烈,但与细胞生长状态的相关关系不够明显,其特异性不高使其不能作为理想的指示细胞原位生长率的指标.     

用扫描电镜观察裸甲藻的方法研究

将多聚甲醛固定的裸甲藻(Takayama pulchellum,TPXM)细胞采用离心法收集,进行小梯度的酒精结合离心法逐级脱水,临界点干燥并真空喷金后,能够在扫描电镜(SEM)下很好地显示出T.pulchellum细胞的完整形态和细微结构,如纵鞭毛、横沟、顶沟、顶突、带状物、藻体线纹、腰鞭毛等;同时环境扫描电镜(ESEM)也能对裸甲藻进行较好的形态学观察,但环境样品的ESEM和SEM电镜观察条件尚需要进一步完善.     

基于18S rDNA序列鉴定北部湾养殖区引发藻华的一株原甲藻

高度保守的核糖体18SrDNA序列可用于经济快捷且精确地鉴定浮游植物的种类。2018年夏季,广西北部湾近海的对虾养殖区内发生了原甲藻引起的藻华,藻华的优势种密度为5.5×105 cells·L~(-1),本文采用形态学观察法结合序列分析鉴定的方法来鉴定该藻的种类。结果表明,在形态上,这株分离培养获得的原甲藻(编号BBGA-9003),其大部分细胞类似心形或卵形,少部分细胞近似三角形,鞭毛2条,细胞长和宽分别为(16.55±1.42)μm和(13.84±1.86)μm。该原甲藻经18SrDNA基因序列测定并进行BLAST分析,结果表明该原甲藻序列和GenBank中56株不同种原甲藻具有很高的同源性,在系统进化树上与不同来源的微小原甲藻聚在一大分支上,且与各微小原甲藻间的遗传距离均小于与其他种间的遗传距离。因此,从分子生物学的角度鉴定该株原甲藻属于微小原甲藻,此种藻华的发生意味着北部湾养殖区有害藻华种类多样性的增加。本研究为18SrDNA序列测定用于海洋浮游植物种类鉴定提供了实验依据,也为广西北部湾养殖区有害藻华的防治提供基础资料。     

实时荧光PCR方法检测含麸质的谷类产品管家基因

采用实时荧光PCR方法鉴别含麸质的谷类成分,选择大麦Hordein管家基因、小麦Gliadin管家基因、黑麦Sec1管家基因和燕麦Avenin管家基因作为物种鉴定的特异性标记基因,设计适合于Taqman探针实时荧光PCR扩增的引物和探针,从而达到鉴别检测食品中含麸质的谷类大麦、小麦、黑麦、燕麦成分的目的.特异性实验结果表明,分别采用大麦、小麦、黑麦、燕麦的引物和探针进行实时PCR扩增时,经检测25种样品,只有相对应的阳性物种样品产生荧光信号,其余非该物种样品均不产生荧光信号,表现出了很高的物种鉴定特异性.以大麦为例的灵敏性实验结果表明,原料样品检测灵敏性可达到0.01%,加工后的食品则可达到0.05%,符合食品成分痕量检测要求.     

实时荧光PCR技术检测致敏原小麦的研究

采用实时荧光PCR技术检测致敏原小麦成分.试验结果表明:采用小麦管家基因GAG56D和Wx012基因的特异性检测引物和探针进行检测时,26种样品经实时PCR扩增,只有小麦产生阳性的荧光信号,其余样品均不产生荧光信号.灵敏度试验结果表明:该方法对小麦致敏原基因的检测灵敏度较高,可达到1 mg.kg-1,符合痕量检测要求.     

基于氧化石墨烯构建新型荧光生物传感器用于DNA检测

以氧化石墨烯作为荧光猝灭基底,羧基荧光素标记的DNA探针作为识别元件,设计了一种用于检测单链DNA的新型荧光生物传感器,通过测定荧光探针分子和目标DNA作用前后体系的荧光强度变化实现特定序列单链DNA的定量检测.在该方法中,荧光强度恢复值与目标DNA浓度在20～1 000 pM范围呈线性关系,检测限为0.11 pM.该方法灵敏度高,操作简单,在生物分子检测及疾病早期诊断中具有较大的应用前景.     

基于单链DNA-银纳米簇荧光探针对微囊藻毒素-LR的检测研究

以单链DNA为模板,制备了单链DNA-银纳米簇(ssDNA-Ag NCs)荧光探针,构建了一种无酶无标记检测微囊藻毒素-LR(microcystin-LR)的荧光传感分析方法.设计的ssDNA既能作为模板合成ssDNA-Ag NCs荧光探针,又能与目标分析物微囊藻毒素-LR通过高亲和性和高特异性结合.采用透射电镜(TE...     

两种赤潮藻对温度、氮、磷营养的反应及活体荧光特性的研究

在实验室分批培养条件下,研究了温度及氮磷营养元素对两种赤潮藻增殖的影响,发现海洋原甲藻(Prorocentrum micans)要求的温度较低,其生长最适温度为23℃,生长温度上限在33℃至34℃之间;微小角毛藻(Chaetoceras minutissmus)的生长最适温度约为30℃,生长温度上限在41℃与44℃之间。氮磷缺乏使两种藻的增殖受阻,生长周期缩短,且缺氮比缺磷的影响大,测定表明两种藻的荧光激发光谱的高峰位置都在580nm附近,而荧光发射光谱的峰位为675nm.DCMU能引起两种藻活体荧光的增益,海洋原甲藻的增益荧光比率(F_D/F_X)为2.5,角毛藻的荧光比率较低,通常小于2.0。     

四种赤潮藻多克隆抗体的制备及特异性分析

为了建立4种赤潮藻（赤潮异弯藻（Heterosigma akashiwo）、海洋褐胞藻（Chattonella,marina）、卵圆褐胞藻（Chattonella ovata）、具齿原甲藻（Prorocenlnmz dentatum））的免疫学检测方法,分别用4种赤潮藻免疫Balb／C小鼠,制备针对4种不同赤潮藻的抗血清,间接ELISA检测其亲和性和特异性．结果显示,4种赤潮藻的抗血清对各自藻体均有良好的亲和力和特异性,交叉反应结果显示,抗血清与同种藻之间有较强的反应而异种赤潮藻之间交又反应较弱．表明单种藻免疫获得的小鼠多克隆抗体可特异性地与相应赤潮藻作用,可用于赤潮藻的免疫学检测分析．     

利用粳稻基因组DNA和C_ot-1 DNA探针对普通野生稻和亚洲栽培稻的比较分析

以粳稻日本晴基因组DNA和Cot-1 DNA为探针,分别对日本晴、籼稻广陆矮4号和普通野生稻的染色体组进行了基因组原位杂交(GISH)和Cot-1 DNA荧光原位杂交(FISH)分析,并对3种染色体组进行了同源聚类和比较研究.结果表明:粳稻基因组DNA和Cot-1 DNA探针信号在3种水稻染色体组中的分布状况和覆盖率相似,Cot-1 DNA的覆盖率分别为(47.13±0.18)%、(45.89±0.22)%、(44.24±0.21)%,3种水稻基因组同源性高,亲缘关系接近.Cot-1 DNA在3种水稻染色体上的杂交信号分布各有特点,中高度重复序列的变异在普通野生稻向栽培稻进化和亚洲栽培稻籼、粳分化过程中具有重要意义,中高度重复序列含量较低的2、5、8号染色体是水稻染色体组进化过程中相对活跃的成分.     

LA-PCR法制备赤麂Y2涂染探针与原位杂交

应用显微切割技术获得赤麂的Y2单条染色体,经LA-PCR（linker-adaptor PCR）扩增后用DIG标记制备探针,然后用Southern blot杂交法对所制备的探针进行验证并对毛冠鹿的中期核型进行原位杂交.结果表明：用该法制备的涂染探针是成功的,原位杂交也获得了阳性结果,可以初步验证毛冠鹿中的Y染色体.     

一种改进的重复多色荧光原位杂交技术

多色荧光原位杂交技术是在常规荧光原位杂交技术基础上建立的一种染色体分析手段,已广泛应用于肿瘤遗传学的研究,尤其是用于染色体隐匿性重排及复杂核型分析。本研究旨在利用现有实验条件,建立一种可靠的人类全染色体分析方法——重复多色荧光原位杂交技术（repetitive multicolour fluorescence in situ hybridization,RM-FISH）。采用特异的全染色体涂染探针,分别用C3、Cy5、FITC3种荧光素直接标记,组合制备4组六色的探针池。对同一染色体片先后进行4次杂交,通过装备了4组滤镜的荧光显微镜捕获信号。RM-FISH技术结合反相DAPI显带对1例原发性食管鳞癌进行核型分析,获得了全部染色体的特异信号。本研究建立的RM-FISH简便、易行,是光谱核型分析的一种较理想的替代技术。     

对于基因芯片实验的动力学范围和结果可重复性的研究

杂交动力学和实验结果的可重复性是基因芯片研究中十分重要的内容，以单基因模拟芯片对DNA芯片的杂交动力学进行了初步的研究：将不同含量的植物cDNA点在玻片上制成DNA芯片，利用体外转录的方法得到植物基因的mRNA，然后通过反转录方法把荧光标记到cDNA上，用不同含量的荧光标记cDNA对芯片进行杂交，根据结果分析了靶基因和探针含量变化对芯片杂交信号产生的影响，利用多基因模拟芯片分析了不同基因的杂交动力学特点；利用12800点的高密度芯片进行了2组不同的表达谱实验，对芯片结果的可重复性做了验证。     

A marine microbial consortium apparently mediating anaerobic oxidation of methane

A large fraction of globally produced methane is converted to CO2 by anaerobic oxidation in marine sediments. Strong geochemical evidence for net methane consumption in anoxic sediments is based on methane profiles, radiotracer experiments and stable carbon isotope data. But the elusive microorganisms mediating this reaction have not yet been isolated, and the pathway of anaerobic oxidation of methane is insufficiently understood. Recent data suggest that certain archaea reverse the process of methanogenesis by interaction with sulphate-reducing bacteria. Here we provide microscopic evidence for a structured consortium of archaea and sulphate-reducing bacteria, which we identified by fluorescence in situ hybridization using specific 16S rRNA-targeted oligonucleotide probes. In this example of a structured archaeal-bacterial symbiosis, the archaea grow in dense aggregates of about 100 cells and are surrounded by sulphate-reducing bacteria. These aggregates were abundant in gas-hydrate-rich sediments with extremely high rates of methane-based sulphate reduction, and apparently mediate anaerobic oxidation of methane.     

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes

Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.     

基于纳米金表面竞争杂交反应的新型DNA检测方法(英文)

基于纳米金表面DNA的竞争杂交反应和纳米金粒子的离心分离-富集过程,建立了一种DNA荧光检测方法,实现对飞摩尔级目标DNA的高灵敏度检测.同时通过DNA的竞争杂交过程并结合缓冲液离子强度的阶跃式升高,实现了对目标DNA上单个碱基错配的有效分辨.本研究还提供了一种简单有效的DNA分离-富集方法,相对于通常使用的磁珠分离过程更为经济方便.     

Discovery of novel intermediate forms redefines the fungal tree of life

Fungi are the principal degraders of biomass in terrestrial ecosystems and establish important interactions with plants and animals. However, our current understanding of fungal evolutionary diversity is incomplete and is based upon species amenable to growth in culture. These culturable fungi are typically yeast or filamentous forms, bound by a rigid cell wall rich in chitin. Evolution of this body plan was thought critical for the success of the Fungi, enabling them to adapt to heterogeneous habitats and live by osmotrophy: extracellular digestion followed by nutrient uptake. Here we investigate the ecology and cell biology of a previously undescribed and highly diverse form of eukaryotic life that branches with the Fungi, using environmental DNA analyses combined with fluorescent detection via DNA probes. This clade is present in numerous ecosystems including soil, freshwater and aquatic sediments. Phylogenetic analyses using multiple ribosomal RNA genes place this clade with Rozella, the putative primary branch of the fungal kingdom. Tyramide signal amplification coupled with group-specific fluorescence in situ hybridization reveals that the target cells are small eukaryotes of 3-5?μm in length, capable of forming a microtubule-based flagellum. Co-staining with cell wall markers demonstrates that representatives from the clade do not produce a chitin-rich cell wall during any of the life cycle stages observed and therefore do not conform to the standard fungal body plan. We name this highly diverse clade the cryptomycota in anticipation of formal classification.     

The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-enhanced piezoelectric biosensor

This paper presents development of a quartz crystal microbalance （QCM） biosensor for real-time detection of E. coil O157：H7 DNA based on nanogold particles amplification. Many inner Au nanoparticles were immobilized onto the thioled surface of the Au electrode, then more specific thiolated sin- gle-stranded DNA （ssDNA） probes could be fixed through Au-SH bonding. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA of E. coli O157：H7 gene eaeA, then resulted in a mass change and corresponding frequency shifts （ △f ） of the QCM. The outer avidin-coated Au nanoparticles could combine with the target DNA to increase the mass. The electrochemical techniques, cyclic voltammetry （CV） and electrochemical impedance spectroscopy （EIS） were adopted to manifest and character each step. The target DNA corresponding to 2.0×10^3 colony forming unit （CFU）/mL E. coil O157：H7 cells can be detected by this biosensor, so it is practical to develop a sensitive and effective QCM biosensor for pathogenic bacteria detection based on specific DNA analysis. The piezoelectric biosensing system has potential for further applications, such as food safety and environment monitoring, and this approach lays the groundwork for incorporating the method into an integrated system for in-field bacteria detection.     

Investigation of self-assembled protein dimers through an artificial ion channel for DNA sensing

Artificial nanopores have become promising tools for sensing DNA. Here, we report a new technique for sensing DNA through a conical-shaped nanopore embed- ded in track-etched polyethylene terephthalate （PET） membrane. Two different streptavidin-conjugated mono- valent DNA probes were prepared that can bind to two distinct segments （at either end） of the target DNA. The size of target DNA-linked to the two streptavidin-conju- gated monovalent DNA probes is double that of the indi- vidual probes. By precisely controlling the tip diameter of the conical nanopore embedded in the PET polymer, events due to the translocation of the streptavidin-conjugated monovalent DNA probes through the nanopore can be fil- tered and purposely undetected, whereas the current pulses due to the translocation of the target DNA-induced self- assembled complexes can be detected. The two streptavi- din-conjugated DNA probes cannot be linked by multi- mismatched DNA. Therefore, multi-mismatched （non- specific） DNA will not induce any current pulse signatures. The current pulse signatures for the self-assembled com- plex can be used to confirm the presence of the target DNA. The size-dependent detection of self-assembled complexes on the molecular level shows strong promise for the detection of biomolecules without interference from the probes.