肽核酸探针技术在赤潮生物检测中的应用

国家重点基础研究发展项目973(2001CB409704);; 863计划(2003AA635060);; 2004年度厦门市科技创新基金项目(3502Z20041059);; 福建省海洋与渔业局科技项

赤潮生物毒素的药理作用研究进展

20 0 4年度厦门市科技创新基金项目 ;; 2 0 0 4年福建省海洋与渔业局科技项目 ;; 国家自然科学基金 (40 0 760 31 ;40 3760 32 )资

Comparative Study on Method of Amplifying the rRNA of the Genomes from Gymnodinium sp. and Prorocentrum minimum by PCR

以赤潮种裸甲藻(Gymnodiniumsp.)和微小原甲藻(Prorocentrumminimum)为试验材料,以不同方法提取 其基因组并进行纯化,然后采用PCR方法扩增其rRNA基因,包括18S,28S和ITS片断,并进行扩增条件的比较和优化,得到两种藻的最佳DNA提取条件和PCR扩增条件.裸甲藻和微小原甲藻的DNA提取宜采用改良的CTAB 方法;并需对粗提取的DNA用CTAB方法进行纯化.两种藻的最适模板浓度为纯化后模板1 0～2.0μL;最适Mg2+ 浓度为2 0μL(25mmol/L);ITS引物PCR扩增的退火温度为50℃,而18S三对引物的退火温度均为55℃,28S的退 火温度为54℃最为适宜.The genomes from bloom-forming species, Gymnodinium sp.and Prorocentrum minimum were extracted and purified by a series of methods, and the gene fragments of 18S,28S and ITS (Internal Transcribed Spacer) of Gymnodinium sp.and Prorocentrum minimum genomic DNA were amplified by polymerase chain reaction(PCR).All of the extracting methods and PCR condition were optimized by comparing its advantages, and the preferable methodological conditions were achieved. The advisable method of extracting and purifying genome was the improved method of CTAB, and the genomes should be purified by CTAB also.The better concentration of DNA template was 1.0～2.0μL of purified genomes from Gymnodinium sp.and Prorocentrum minimum, and the concentration of Mg~(2+) was 2.0μL of 25 mmol/L in 25μL PCR systems.The advisable annealing temperature was 50℃ to primer pair of ITS, 55℃ to 18S, and 54℃ to 28S respectively in PCR program.国家重点基础研究发展规划973项目(G1999043706);; 国家自然科学基金(40076031;40376032)资助项目

化学分类法在有害水华生物分类鉴定中的应用

在综合分析有害水华生物分类鉴定方法研究概况的基础上,阐述了基于光合色素和其它生化标志物的化学分类方法在水华生物检测和鉴定方面的应用,讨论了基于叶绿素a成分分析的监测鉴定技术和基于色素荧光分析的流式细胞技术(FCM)在水华生物化学分类上的优势和特点。相关的分析表明化学分类法能有效地用于对不同属的有害水华生物进行分类鉴定,甚至也能对相同属和种的不同细胞株进行鉴定识别,从而对有害水华生物的鉴定提供较为丰富的分类信息

MOLECULAR TAXONOMY ANALYSES OF THE RELATIONSHIPS IN SEVERAL REPRESENTATIVE HARMFUL ALGAL BLOOM SPECIES

通过PCR克隆测序、rDNA序列分析、随机PCR引物扩增结合DGGE技术等三个层次的分子分类水平对厦门海域的典型藻华生物进行分子生物学分析。结果表明,基于DGGE检测的18S rDNA序列过于保守,分类的精确度不高;AP-PCR则是基于基因组的差异进行分析,结果较为精确和全面;而rDNA序列分析相对可靠,尤其是针对ITS的长度和全序列分析以及28S rDNA的D1、D2区的序列分析,可以提供更为准确的分类信息,并可为设计检测探针提供基础。据此对分离自厦门海域的3种典型甲藻及其相关藻株建立了系统进化树。针对23株甲藻ITS序列建立的系统进化树显示Takayama pulchella(AY764179)和Karlodinium micrum的距离最近,并能够把Akashiwo属、Karenia属、Gyrodinium属、Karlodinium属、Takayama属与Gymnodinium属等区分开。用28S rDNA序列建立的系统发育树只能把Karlodinium属、Karenia属和Takayama属区分开,但不能很好地把无纹环沟藻与Akashiwo属和Gymnodinium等的藻区分开... 【英文摘要】 With rapid economic development and increasing pollution,some Chinese coastal waters have become eutrophic,which has resulted in a high frequency of harmful algal blooms.Rapid and unequivocal identification of harmful species has become a focal point of recent toxic algal research.At present,morphological criteria are the primary means to identify and classify harmful algae,but it is often difficult to distinguish morphologically-similar species and differentiate non-toxic from toxic algae.Some new molecula...国家重点基础研究发展规划973项目(编号:CEOHAB2001CB409704);; 中国博士后基金项目(编号:20060400854);; 教育部博士点基金项目(编号:20070504076);; 淡水生态与生物技术国家重点实验室开放课题(编号:2008FB005);; 农业部淡水鱼类种质资源与生物技术重点实验室开放课题(编号:LFB20070611)资

STUDY ON DETECTION OF IN SITU GROWTH RATE OF TAKAYAMA PULCHELLUM

HOU Jian-Jun1; 2; 3; 4; LAI Hong-Yan1; LEI Hong-Ling3and HUANG Bang-Qin2（1.College of Fisheries; Huazhong Agricultural University; Wuhan 430070; 2.State Key Laboratory of Marine Environmental Science; Xiamen University; Xiamen 361005; 3.Key Laboratory of Biologic Resources Protection and Utilization of Hubei Province（Hubei Institute for Nationalities）; Enshi 445000; 4.Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology（Yangtse River Fisheries Research Institute; Chinese Academy of Fishery Sciences）; ...;国家重点基础研究发展规划973项目(编号:CEOHAB2001CB409704);; 农业部淡水鱼类种质资源与生物技术重点实验室开放课题(编号:LFB20070611);; 中国博士后基金项目(编号:20060400854);; 教育部博士点,基金项目(编号:20070504076)资

Detection of representative harmful algae using three kinds of molecular probes

集中使用寡核苷酸、肽核酸和细胞凝素3类探针对来自东海和厦门海域的现场赤潮样品进行了检测,尝试鉴定识别自然水样中有害的赤潮原因种塔玛亚历山大藻,微小原甲藻和纤小裸甲藻,建立和优化了这些探针的检测方法和样品处理程序。结果表明,在东海和厦门海域的赤潮样品中均成功地检出了塔玛亚历山大藻的分布情况,各探针的检测效率为DBA>Tama28S>Tama5S;在东海和厦门海域的赤潮样品中,也成功地检测出了微小原甲藻,各探针的检测效率为:ConA>PM18S02>PM28S02;在厦门海域的赤潮水样中检出了纤小裸甲藻,各探针的检测效率为:WGA>PNATP28S01>TP18S02>TP28S01。各探针检测结果与相关文献的报道吻合较好。比较这3类探针的特异性,其中以PNA探针为最好,其次为DNA;lectin探针的特异性相对较弱。 【英文摘要】 Molecular probes are useful to separate closely related harmful algal bloom species. Moreover, they can rapidly and clearly differentiate non-toxic from toxic algae and to monitor the development of algal blooms in coastal waters. There are three kinds of molecular probes which can be used for identification of HAB species, such as lectin, DNA and peptide nucleotide acid(PNA) probe. These probes are relatively new, and have higher sensitivity, veracity and specificity than traditional taxonomic techniques. ...国家“九七三”重点基础研究发展规划(CEOHAB2001CB409704);; 中国博士后基金(20060400854);; 农业部淡水鱼类种质资源与生物技术重点实验室开放课题(LFB20070611);; 厦门市创新科技基金(3502Z20041059);; 教育部博士点基金(20070504076

Preparation of single molecular film of protein A to assemble antibody for construction of biomarker chip

免疫分析方法主要依赖于固定在固相基底上的抗体对相应抗原的特异性识别,抗体在固相基底上固定后最大程度保持生物学活性是设计免疫分析方法的关键技术.本研究利用在疏水性硅基底表面制备蛋白A单分子膜层可以特异结合抗体的Fc端,进而实现对抗体的定向化装配,进一步构建格式化阵列用于肿瘤标志物检测.研究结果表明,制备的蛋白A单分子膜层的厚度为1.8±0.6nm,抗体可以经蛋白A单分子膜层实现定向化装配,由此设计的传感阵列检测肿瘤标志物甲胎蛋白(alpha-fetoprotein,AFP)可以实现1.0ng/mL的灵敏度,血清检测结果与电化学法(ECLIA)测定进行统计学检验没有显著性差异(P>0.05)

Observation of harmful algal blooms caused by Prorocentrum and Gymnodinium species in the western Xiamen Harbour

对近年来发生在厦门海域的裸甲藻(Gymnodinuum)和原甲藻(Prorocentrum)赤潮的发生情况进行了监测分析,采用了采样、分离、单种培养、显微镜和扫描电镜观察r、DNA序列分析等系列监测、分离培养和赤潮生物鉴定技术,重点观察并确证了厦门海域存在的赤潮原因种为微小原甲藻(Prorocentrum minimum)、Takayama pulchellum、无纹环沟藻(Gyrodinium instriatum)。光学显微镜观察表明,赤潮发生海域存在着许多原甲藻和裸甲藻种类,但不能进一步确认到种。利用电子显微镜观察,可根据微小原甲藻体表规则的花纹等特征,根据Takayama pulchellum具有明显的特异性反S形顶沟等特征分别对它们进行有效地分类鉴定。分子分类学分析表明,T.pulchellum(株名为TPXM)28S rDNA D1-D2区序列长度为721 bp,与基因库中同种相似株的同源性大于99%;微小原甲藻(株名PMDH)的ITS和28S rDNA序列与基因库中同种序列的同源性高达99%;无纹环沟藻(株名GIXM)的ITS与基因库中登记的分离自中国深圳海域的4株同种藻的同源性也高达99%。用ITS序列和28S rDNA序列建立的系统进化树也能很好地显示微小原甲藻、Takayama pulchellum、无纹环沟藻之间以及它们与其他藻之间的亲缘关系。将上述结果结合文献记录和环境条件进行了分析,证实这3种赤潮种类Takayama pulchellum、无纹环沟藻、微小原甲藻是厦门海域较为常见的赤潮原因种。对上述检测和鉴定方法的系统应用也表明,这些方法可应用于对现场赤潮生物进行有效监测。Harmful algal blooms(HABs) is increasing globally,and HABs is one of the major disasters along the coast of Xiamen Harbour.Further investigations and researches on the main types of HABs and the algal species causing HAB in coast water of Xiamen Harbour should be made;the environmental monitoring networks and methods along the coast of Xiamen Harbour should be set up and consummated;science researches with local characteristics should be fully developed,and the exotic HAB species from the cabin seawater of foreign ships should also be studied.Aiming directly at rapid identification and regular monitoring of HABs species,this paper tried to develop essential methods of identificating and monitoring HABs species,and evaluated systemic methods for rapidly monitoring and classifying HABs species in morphology and molecular taxonomy.Meanwhile,series of studies were also undertaken to evaluate in the field and tried to find these important HABs causing species along the coast water of western Xiamen Harbour.The harmful algal bloom events caused by Prorocentrum and Gymnodinium species in coast water of western Xiamen Harbour were monitored during recent years.A series of detecting methods based on the analysis of environmental factors,sampling,cell isolation and unicellular culture of these HABs species,observation under microscope and scanning electron microscope(SEM),and sequence analysis of rDNA were used together to monitor and identify these harmful algae.The methods used for collecting samples and establishing specific HAB unicellular cultures were described in detail.Methods of SEM(scanning electron microscope) and ESEM(environmental scanning electron microscope) for Gymnodinium were developed and optimized.These important species that caused harmful algal bloom in Xiamen Harbour were observed and identified as Takayama pulchellum(strain name:TPXM),Gyrodinium instriatum(strain name:GIXM) and Prorocentrum minimum(strain name:PMXM).Algal morphology was observed under LM,and the results suggested that there were many Prorocentrum and Gymnodinium species in coast water of western Xiamen Harbour,and these species could not be identified as specific species under LM.Two strain of Prorocentrum and Gymnodinium were isolated during blooms in Xiamen Harbor and their morphology were observed under SEM.The results showed that there were regular patterns and other characteristics on Prorocentrum cells,which indicated that PMXM was Prorocentrum minimum,and cells of TPXM were broadly oval with a conspicuous and well-defined sigmoid apical groove present on the epitheca,and the apical groove was a characteristic reversed S-shape.These characteristics showed that TPXM was Takayama pulchellum.Molecular biological analyses of rDNA sequences of Prorocentrum and Gymnodinium showed that the D1-D2 region of partial LSU(large subunit) rDNA of T.pulchellum(TPXM) had 721bp and shared more than 99% similarity to the same species whose data was deposited in the GenBank,the ITS and D1-D2 region of partial LSU of P.minimum(PMXM) also shared more than 99% similarity to the same species in the GenBank,and ITS region of G.instriatum(GIXM) shared more than 99% similarity to the same species isolated from Shenzhen Bay in China and its sequences were deposited in the GenBank,too.The analysis of rDNA sequences and phylogenetic trees could be an appropriate taxonomic evidence,especially the length and whole sequences analysis of ITS and variable regions D1-D2 of 28S rDNA could be more reliable and could provide more exact taxonomic informations than other methods.The phylogenetic trees constructed from ITS and partial 28S rDNA could show the relationships of T.pulchellum(TPXM),G.instriatum(GIXM) and P.minimum(PMXM) and their correlative species,and could also separate these species from their disrelated species clearly.According to above results,the characteristics and causation of HABs in western Xiamen Harbour caused by the three species were discussed based on the analysis of related references and environmental conditions.All the results suggested that the three HABs causing species,such as T.pulchellum(TPXM),G.instriatum(GIXM),P.minimum (PMXM) were common HAB species in coast water of western Xiamen Harbour.The results of methodological application in field indicated that these methods and protocols might become useful,feasible and reliable tools to monitor in situ distribution of bloom-forming taxa,such as Gymnodinium and Prorocentrum in natural water.国家重点基础研究发展规划973项目(CEOHAB2001CB409704);; 厦门市创新科技基金项目(3502Z20041059);; 中国博士后基金项目(20060400854);; 农业部淡水鱼类种质资源与生物技术重点实验室开放课题(LFB20070611

制备蛋白A单分子膜层装配抗体构建肿瘤标志物检测芯片

免疫分析方法主要依赖于固定在固相基底上的抗体对相应抗原的特异性识别,抗体在固相基底上固定后最大程度保持生物学活性是设计免疫分析方法的关键技术.本研究利用在疏水性硅基底表面制备蛋白A单分子膜层可以特异结合抗体的Fc端,进而实现对抗体的定向化装配,进一步构建格式化阵列用于肿瘤标志物检测.研究结果表明,制备的蛋白A单分子膜层的厚度为1.8±0.6nm,抗体可以经蛋白A单分子膜层实现定向化装配,由此设计的传感阵列检测肿瘤标志物甲胎蛋白(alpha-fetoprotein,AFP)可以实现1.0ng/mL的灵敏度,血清检测结果与电化学法(ECLIA)测定进行统计学检验没有显著性差异(P>0.05)