应用荧光原位杂交方法检测亚历山大藻

采用PCR方法对三株亚历山大藻核糖体DNA 大亚基部分序列和ITS区序列进行了特异性扩增和序列测定.ITS区测序及BLAST比对结果显示,三株藻类分别为塔玛亚历山大藻(Alexandrium tamarense),链状亚历山大藻(A. catenella)和微小亚历山大藻(A. minimum).在此基础上,针对中国沿海分布的有毒亚历山大藻,根据其核糖体DNA大亚基(LSU rDNA)序列信息,设计了特异性的荧光标记探针,建立了基于荧光原位杂交技术(FISH)上的有毒亚历山大藻检测方法.结果表明,探针Alexp1较理想地标记选定的目标藻--塔玛亚历山大藻(YA 藻株),经探针标记的藻细胞在荧光显微镜下可以明显区分于其他非目标藻.     

应用荧光原位杂交方法检测赤潮异弯藻

根据对赤潮异弯藻(Heterosigma akashiwo)核糖体大亚基(LSU)和核糖体转录单元内间隔区 (ITS) 序列信息的分析,设计了以胞质rRNA和胞核rDNA为靶序列的特异性探针,建立了赤潮异弯藻的全细胞和细胞核荧光原位杂交快速检测方法.结果表明,探针能分别使整个细胞质和细胞核呈现强烈的绿色荧光;探针是特异性的,不与其他受试藻进行交叉反应.同时,用该方法成功的从模拟自然海区混合样品中检测出赤潮异弯藻.该研究方法可以适用于对实验室和自然海区赤潮异弯藻样品的快速、准确、特异和半定量检测.     

塔玛亚历山大藻溶藻细菌筛选方法的初步研究

旨在对溶藻细菌筛选方法的摸索.从辽宁大连周边海域分离的37株海洋细菌中,尝试用试管法筛选对塔玛亚历山大藻具有溶藻能力的功能细菌.试管法即首先在试管中进行藻菌共培养,待初步筛选出合适菌株后,再用三角瓶来验证初筛结果.结果显示:试管初筛选出的5株溶藻菌株,同样在三角瓶中显示了它们溶藻能力,证明试管法用于筛选溶藻细菌是可行的.     

应用qPCR方法检测中国近海塔玛亚历山大藻复合种的研究

亚历山大藻属的部分藻种(Alexandrium spp.)能够产生麻痹性贝毒毒素(PST),是重要的有害藻华(HAB)原因种.由于亚历山大藻属中的有毒和无毒藻种形态相似,且海水中亚历山大藻的细胞密度通常很低,因此,高灵敏度、高特异性的分子生物学方法在亚历山大藻检测方面具有重要的应用前景.塔玛亚历山大藻和链状亚历山大藻是中国近海主要的有毒亚历山大藻藻种,大多属于塔玛亚历山大藻复合种第四类核糖体型(GroupⅣ,以往称为“亚洲温带核糖体型”).因此,本研究尝试将实时荧光定量PCR(qPCR)方法用于我国近海塔玛亚历山大藻复合种(GroupⅣ)的快速检测,并对方法的特异性和灵敏度进行了检验.研究表明,所建立的qPCR方法能够特异性地检测我国近海不同海域分离的塔玛亚历山大藻复合种(GroupⅣ),方法具有良好的线性响应关系和较高的灵敏度,最低可检出5个藻细胞,具有良好的应用前景.然而,实验也发现,自我国近海分离的塔玛亚历山大藻复合种的不同藻株之间,以及处于不同生长阶段的藻细胞之间,qPCR检测结果存在显著差异,反映了目标藻核糖体RNA基因拷贝数的差异和变化对qPCR检测结果的影响.因此,建议在将qPCR方法用于不同海域亚历山大藻样品检测时,应采用该海域分离的藻株专门构建标准工作曲线,以减小定量分析误差.     

海洋产毒甲藻——塔玛亚历山大藻(Alexandrium tarmarense)基因组DNA微卫星位点分离

采用FIASCO(Fast Isolation by AFLP of Sequences Containing repeats site)方法富集、分离海洋产毒甲藻——塔玛亚历山大藻基因组DNA微卫星位点。结果发现,测序分析的22个克隆中全部含有微卫星位点,插入片段大小为175-687 bp,微卫星DNA的重复数最低4次,最高27次,平均11次;在全部的39个微卫星位点中,其中25个属于完美重复型,13个不完美重复型,1个复合型。为塔玛亚历山大藻系统进化生物学、分子生态学研究提供了很好的标记位点。     

氮磷比对蛋白核小球藻和塔玛亚历山大藻种间竞争的影响

通过单独培养和共同培养的方法,以Na NO3和KH2PO3为氮源和磷源,研究了氮磷比(4、6、9、10、13、16、22、36和91)对蛋白核小球藻(Chlorella pyrenoidosa)和塔玛亚历山大藻(Alexandrium tamarense)生长和种间竞争的影响,并对其作用机制进行了探讨.结果表明:在单独培养模式下,蛋白核小球藻在N/P为4~22时生长适宜,N/P22时生长受抑制,而塔玛亚历山大藻在N/P为4和6时生长适宜,N/P6时生长受抑制;在共同培养模式下,蛋白核小球藻具有竞争优势,而塔玛亚历山大藻的生长受抑制,且N/P为13和16时蛋白核小球藻种群竞争优势最明显;蛋白核小球藻和塔玛亚历山大藻的培养滤液对彼此生长均未产生抑制作用,由此推测,两种微藻的种间竞争主要以资源利用性竞争为主.结果可为开发利用饵料藻进行赤潮生物防控提供一些理论依据.     

赤潮过程中塔玛亚历山大藻(Alexandrium tamareme)C、N稳定同位素组成的变化规律

为了探索赤潮监测预防的新思路和方法,本研究选取典型赤潮藻(塔玛亚历山大藻Alexandrium tamareme)作为实验藻种,模拟一次完整的赤潮形成过程,测定重要监测指标(生物量、营养盐浓度)的变化,找出稳定同位素特征值与之相关变化规律。实验结果表明,藻种生长过程可明显分为四个阶段:适应期、对数生长期、稳定生长期及衰亡期;N、P浓度随培养时间呈下降趋势,与生物量的变化有很好的相关性,但在生长过程中几乎不吸收硅酸盐。在一次完整的赤潮发生过程中,C稳定同位素组成(13C值)随时间显示出减小趋势,而N稳定同位素组成(15N值)值则显示增大趋势。因此,赤潮藻的C和N稳定同位素组成为赤潮监测提供了可能的新指标和新方法。     

两种抗生素对塔玛亚历山大藻生长和产毒的影响

论文以中国南海筛选的一株塔玛亚历山大藻Alexandrium tamarense C101为研究对象,研究了两种抗生素,氨苄青霉素(ampicillin,Amp)和新霉素(neomycin,Nm)对A.tamarense C101细胞生长、形态以及毒素含量的影响.结果表明:和空白对照组相比,经1 500μg/mL Nm...     

单种及混合培养条件下Fe、Mn对赤潮生物塔玛亚历山大藻(Alexandriumtamarense)生长的影响

研究了单种和两种混合世量培养条件下，Ｆｅ－ＥＤＴＡ（Ｆｅ＾３＋）、Ｍｎ（Ｍｎ＾２＋）浓度对甲藻塔玛亚历山大藻（Ａｌｅｘａｎｄｒｉｕｍ ｔａｍａｒｅｎｓｅ）生长及细胞大小的影响。同时考察了与其混合培养中的肋骨条藻（Ｓｋｅｌｅｔｏｎｅｍａ ｃｏｓｔａｔｕｍ）和圆筛藻（Ｃｏｓｃｉｍｏｄｉｓ ｃｕｓ ｓｐ．）的生长特性，结果表明在本实验的浓度范围内（Ｆｅ－ＥＤＴＡ，０－３．１５μｇ／Ｌ，Ｍｎ＾２＋，０－０     

桉木粉对塔玛亚历山大藻（Alexandrium tamarense）的抑制作用及其化学基础研究

摘要：探讨了托里桉(Eucalyptus torelliana)、尾叶桉(Eucalyptus urophylla)、窿缘桉(Eucalyptus exserta)等3种桉木粉对塔玛亚历山大藻（Alexandrium tamarense）生长的影响,并对其抑制赤潮藻类生长的化学基础进行了初步研究,以期为新除藻剂的筛选提供参考和依据。结果表明,不同桉木粉对塔玛亚历山大藻生长的抑制作用不同,托里桉木粉的抑藻作用明显强于尾叶桉和窿缘桉。灭菌与非灭菌托里桉木粉对塔玛亚历山大藻生长的抑制作用差别不大,提示细菌等微生物并非木粉抑藻的主要原因。不同溶剂粗提物对塔玛亚历山大藻生长的抑制作用不同,丙酮-水粗提物的抑藻活性明显强于乙酸乙酯、甲醇和水粗提物。进一步将丙酮-水粗提物分成A、B、C和D等4个组分,比较不同组分的抑藻活性。结果发现,丙酮-水粗提物中D组分的抑藻作用最强。该组分浓度为3 mg/L时,3 d时对塔玛亚历山大藻的去除率可达81.06 %。GC-MS分析表明,D组分中含有4-羟基-3,5,6-三甲基,1-4[(1z)-3-氧代-1-丁烯基]-2-环已烯酮和4,4,7a–三甲基,5,6,7,7a-四氢-2(4H)-苯并呋喃酮等酮类物质。这些结果表明,托里桉木粉可显著抑制赤潮藻的生长,其中存在的酮类化感物质可能是其抑制藻类生长的主要原因。     

荧光原位杂交在环境微生物学中的应用及进展

荧光原位杂交技术广泛用于分析复杂环境的微生物群落构成,可以在自然生境中监测和鉴定微生物,并能对未被培养的微生物进行检测.rRNA为靶序列寡核苷酸或PNA探针的荧光原位杂交能提供微生物的形态学、数量、空间分布等信息,现已成为环境科学研究领域中的热点技术.对荧光原位杂交技术的发展和在环境微生物学中的应用进行了综述,探讨了该技术的应用和发展前景.      

Fluorescence in situ hybridization (FISH) for rapid detection of aneuploidy: experience in 911 prenatal cases†

Fluorescence in situ hybridization (FISH) was performed with probes specific for chromosomes 13, 18, 21, X and Y on 911 of 11123 (8.2%) amniotic fluid samples submitted to the present authors' laboratory for cytogenetic analysis over an 8-year period. Altogether 3516 hybridizations were performed with an interpretable FISH result on all chromosomes requested in 884/911 (97%) of cases. An uninformative FISH result occurred in 44 hybridizations among 27 cases (3%). Of a total of 89 karyotypically proven cases with aneuploidy that might have been detected by FISH, the overall detection rate was 84%. An inconclusive or incomplete FISH result occurred in 9/89 (10%) of these proven aneuploid cases. In the remaining 80 informative proven aneuploid cases, correct detection of aneuploidy was accomplished in 75/80 (94%) of samples. A false-negative result occurred in the remaining 5/80 (6%) of such informative cases. Eighteen cases had karyotypically proven abnormalities that could not have been detected by the targeted FISH. Aside from these 18 cases, FISH allowed correct detection of normal disomy in 785/804 (98%) of such cases. An incomplete FISH result occurred in 18 normal disomic cases. There was a single possible ‘false-positive’ FISH result for chromosome 21. Interphase FISH analysis of uncultured amniotic fluid cells has been shown to be a useful laboratory tool for rapid fetal aneuploidy screening during pregnancy. As with all clinical laboratory diagnostic tests, incomplete or inconclusive results (or even interpretive errors) occur in a small percentage of cases. Nevertheless, FISH results accompanied by other data and by appropriate counseling provide clinicians and patients with valuable information for clinical decision-making surrounding family planning and pregnancy management. Copyright © 2001 John Wiley & Sons, Ltd.     

荧光原位杂交法在活性污泥硝化细菌检测中的应用

采用荧光原位杂交(FISH)法对活性污泥样品中的硝化细菌进行了检测研究。与传统方法相比,FISH法具有快速便捷的特点,可原位检测出活性污泥菌胶团上的硝化细菌的分布情况,结合稀释的方法,可对环境样品中的硝化细菌进行定量分析。此法既可定性,又可对环境中的硝化细菌进行直接计数,为研究硝化细菌类群的空间和数量分布提供有效的检测工具。     

Magnetic-activated cell sorting (MACS) significantly decreases the hybridization efficiency of fluorescence in situ hybridization (FISH)

Fetal cells were enriched from maternal blood using density gradient centrifugation of Histopaque followed by magnetic-activated cell sorting (MACS) to select CD71-positive cells. For each specimen, cells partially purified by Histopaque were split into equal portions, and each portion was subjected to purification by MACS in parallel. Cells before and after MACS were subjected to dual-color fluorescence in situ hybridization (FISH) analysis with X- and Y-chromosome-specific probes. We found that the hybridization rates were decreased by approximately 10% after MACS based on duplicated analysis for each sample. Copyright © 2001 John Wiley & Sons, Ltd.     

Distribution characteristics of ammonia-oxidizing bacteria in the Typha latifolia constructed wetlands using fluorescent in situ hybridization(FISH)

A molecular biology method, fluorescent in situ hybridization（FISH）, in which the pre-treatment was improved in allusion to the media of the constructed wetlands（CW）, e.g. the soil and the grit, was used to investigate the vertical distribution characteristics of ammonia-oxidizing bacteria（AOB） quantity and the relation with oxidation-reduction potential（ORP） in the Typha latifolia constructed wetlands under three different Ioadings in summer from May to September. Results showed that the quantity of the AOB decreased in the Typha latifolia CW with the increase of vertical depth. However, the AOB quantity was 2-4 times the quantity of the control in the root area. Additionally, ORP in the rhizosphere was found to be higher than other areas, which showed that Typha latifolia CW was in an aerobic state in summer when using simulated non-point sewage at the rural area of Taihu Lake in China and small town combined sewage.     

Prenatal application of fluorescent in situ hybridization (FISH) for identification of a mosaic Y-chromosome marker,IDIC(Yp)

An amniocentesis was performed at 13.3 weeks' gestation for advanced maternal age. A mosaic sex chromosome pattern was found: of 50 cells examined, 34 had a 45,X karyotype. In 14 cells with a modal number of 46, a recognizable Y was substituted by a small non-fluorescent marker. C-banding identified the marker as an isodicentric in 12 cells. In two cells, the non-fluorescent marker appeared to be monocentric and looked like a non-fluorescent del (Yq), but could have been an isodicentric Y with inactivation of one of the centromeres. Two cells with a modal number of 47 showed two copies of the monocentric marker. Fluorescent in situ hybridization with an alpha satellite Y-specific centromeric probe confirmed the Y-chromosome origin of the markers and allowed for more accurate prenatal diagnostic information.     

Molecular cytogenetic analysis of term placentae suspected of mosaicism using fluorescence in situ hybridization

In first-trimester chorionic villus sampling (CVS) for prenatal diagnosis, abnormal chromosomal findings, such as mosaicism, trisomies, or suspect abnormal karyotypes, are found more frequently than at amniocentesis. The fact that these chromosomal abnormalities do not always reflect the fetal karyotype but may be restricted to the placenta is a major problem in diagnosis and counselling. In this paper we present the results of fluorescence in situ hybridization (FISH) studies on interphase nuclei of three term placentae investigated because of false-positive findings at first-trimester CVS. The chorionic villi of the first case showed a mosaic chromosome pattern involving a trisomy 10 cell line and a normal cell line, those of the second case a total trisomy 8 cell line, while in the third case a complete monosomy X was found. Follow-up amniocentesis in each of these three cases revealed a normal karyotype. By using FISH, we were able to confirm the presence of the aberrant cell lines, which were all confined to one part of the placenta. FISH on interphase nuclei allows the investigation of large numbers of cells for the existence of numerical chromosome aberrations in a quick and reliable way.     

FISH法作为厌氧消化系统运行管理指标的可行性

以高浓度厌氧消化污泥为对象,进行FISH、MPN和产甲烷活性的测定实验.FISH分析得到,厌氧消化污泥中古细菌、嗜乙酸产甲烷菌和嗜氢产甲烷菌占全菌数的比例分别为30%～70%,18%～28%和15%～43%,菌数测定结果FISH法高于MPN法.研究表明,FISH法可用于高浓度厌氧消化污泥的菌相解析,作为处理系统运行管理指标使用.     

Swedish survey on extra structurally abnormal chromosomes in 39 105 consecutive prenatal diagnoses: Prevalence and characterization by fluorescence in situ hybridization

During 7 years (1985–1992), 39 105 consecutive prenatal diagnoses (34 908 amniocenteses and 4197 chorionic villus samples) were made at the five largest clinical genetic laboratories in Sweden. Thirty-one cases of extra structurally abnormal chromosomes (ESACs) were found, giving a total prevalence of 0·8 per 1000. Twelve ESACs were inherited, 14 were de novo and in five the parental origin was unknown. This gives an estimated prevalence of 0·3–0·4 per 1000 for familial and 0·4–0·5 per 1000 for de novo ESACs. Retrospectively, the ESACs were characterized by fluorescence in situ hybridization (FISH). In nine cases, no material was available for this analysis. In 21 of the remaining 22 cases, the chromosomal origin could be identified by FISH. Seventeen of these (81 per cent) were derived from the acrocentric chromosomes, of which 13 originated from chromosome 15 (62 per cent). The most common ESAC was the inv dup(15) (57 per cent). Two cases were derived from chromosome 22, one from chromosome 14, and one from either chromosome 13 or chromosome 21. The four remaining cases consisted to two i(18p)s and two small ring chromosomes derived from chromosomes 4 and 19, respectively.