应用双特异分子探针技术定性定量检测圆海链藻(Thalassiosira rotula)

设计了一套圆海链藻(Thalassiosira rotula)特异性探针,运用双特异分子探针技术,对圆海链藻(Thalassiosira rotula)进行了定性定量检测.结果表明,本实验中设计的一套探针与其它十几种藻无交叉反应,具有种特异性;细胞裂解液直接杂交检测优于提纯RNA样品检测;对自然样品做了初步检测,发现天然海水中的其它浮游生物对该检测方法影响很小.     

运用双特异分子探针技术对胶州湾三种硅藻的检测

利用本实验室首创的双特异分子探针技术对胶州湾常见的3种硅藻-旋链角毛藻(Chaeto-ceros curvisetus Cleve)、尖刺拟菱形藻(Pseudo-nitzschia pungens(Grunowex Cleve)Halse)和中肋骨条藻(Skeletonema costatum(Greville)Cleve)进行定性与定量分析。结果表明,无论是对实验室样品还是自然样品进行检测,本技术均有较好的适用性,对3种目标藻的最低检测细胞数分别为4.2×104、9.4×103和6.0×104个细胞。统计分析表明,双特异分子探针技术对3种目标藻的分析结果与经典的显微镜镜检结果没有显著性差异。本技术利用了多孔酶标板进行检测,整个实验过程约7h,可在较短时间内分析大量样品中的多种微藻,为同时监测多种赤潮藻开辟了一条新途径。     

赤潮异弯藻和海洋原甲藻LSU Rdna扩增及序列分析

利用引物D1R和D2C扩增并测定了赤潮异弯藻(Heterosigma akashiwo Hada)和海洋原甲藻(Prorocentrum micans Ehrenberg)的LSU rDNA D1与D2序列,并与GenBank中相关序列进行比较分析.结果表明:在种内水平,所测的H.akashiwo 6个株系之间共有5个变异位点,序列H.k与H.k-2,H.k-4均存在碱基替换;原甲藻属不同种内各株系之间的遗传距离为0.002～0.023之间,所测序列P.mi与P.micans其他株系之间均存在碱基替换.在种间水平上,原甲藻属不同种之间的遗传距离在0.045～0.139之间,大于种内遗传距离,每个种都具有特定的保守序列.根据序列间的遗传变异,可设计特异性的探针对不同株系和物种进行检测和计数.     

东海原甲藻核酸和蛋白对环境中氮磷浓度变化的响应

通过设定不同的氮(N)、磷(P)浓度一次性培养东海原甲藻,研究了该藻的生长状况及不同生长阶段单位细胞DNA,RNA及蛋白质含量的变化,结果表明,不同氮磷浓度对东海原甲藻的生长产生显著影响,最终生物量与初始的氮磷浓度呈正相关,不同培养组的最大生长率存在显著差异(P<0.05);DNA含量与培养液中氮浓度相关性极显著(P<0.01),但与磷浓度相关性不显著(P>0.05);RNA含量与氮磷浓度均呈极显著相关(P<0.01),而且在不同的生长阶段其含量也会发生变化,随着培养时间的延长,各培养组的RNA含量显著下降(P<0.05);RNA/DNA比值与氮磷浓度均显著相关(P<0.05),而且与生长率呈线性相关关系。培养液中氮浓度显著影响到东海原甲藻单位细胞总蛋白含量,磷缺乏也会使总蛋白含量显著降低。     

基于LAMP-LFD技术的有害赤潮藻东海原甲藻(Prorocentrum donghaiense)快检方法

以东海原甲藻的ITS序列(Internal transcribed spacer,ITS)为检测靶标,将生物素标记的环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP)扩增产物与经异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记的探针特异性杂交,并结合横向流动试纸条(Lateral flow dipstick,LFD)肉眼直接观察检测结果,建立了有害赤潮藻东海原甲藻(Prorocentrum donghaiense)的快速检测技术。经优化后的最适条件为63°C、30min,较常规PCR扩增缩短约2h。结果表明:LAMPLFD可特异性检出东海原甲藻,对常见赤潮藻检测结果均为阴性;其对东海原甲藻基因组DNA的检测最低限为47pg/μL,是常规PCR技术(以F3/B3为引物)的10倍。LAMP-LFD技术能高效、特异地检出东海原甲藻,仪器设备依赖性低,结果可视化,有望成为赤潮原因种检测监控的常规方法。     

Detection of Prorocentrum donghaiense using sandwich hybridization integrated with nuclease protection assay

Prorocentrum donghaiense is an important harmful algae bloom (HAB) causing creature in China’s seas, and the conventional visual detection can not cope with long-term monitoring and high-throughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay (NPA-SH) was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, ...     

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified,cloned and sequenced,and these sequence data were deposited in the GenBank.Eight oligonucleotide probes(DNA probes)were designed based on the sequence analysis.The probes were employed to detect and identify P.minimum and T. pulchella in unialgal and mixed algal samples with a fuorescence in situ hybridization method using flow cytometry.Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences,and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe.These DNA probes and the hybridization protocol we developed were specific and effective for P.minimum and T. pulchella,without any specific binding to other algal species.The hyrbridization efficiency of difierent probes specific to P.minimum was in the order:PMl8S02＞PM28S02＞PM28S01＞PM18S01,and that of the probes specific to T. pulchella was TP18S02＞TP28S01＞TP28S02＞TP18S01.The djfferent hybridization efficiency of the DNA probes could also be shown in the fuorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry.The DNA probes PM18S02,PM28S02,TPl8S02 and TP28S01,and the protocol,were also useful for the detection of algae in natural samples.     

Development of taxonomic rRNA-targeted probes of two harmful algae: Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization

Harmful algal blooms recently have been under the spotlight throughout the world, because of their negative impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were taken into consideration. The partial large subunit rDNA (D1-D2) of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization (FISH) tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K. mikimotoi, respectively. Both the specific (taxonomic) (Pmin0443 and Kmik0602) and the control probes (UniC0512 and UniR0499) were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the techniques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P. minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant difference (p >0.05) was observed in the cell densities of the samples determined by FISH and light microscopy (LM). All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.     

Integration of the nuclease protection assay with sandwich hybridization (NPA-SH) for sensitive detection of Heterocapsa triquetra

Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquetra is one of the most common bloom-forming species in estuarine and coastal waters worldwide. Although this species does not produce toxins, unlike some other Heterocapsa species, the high density of its blooms can cause significant ecological damage. We developed a H. triquetra species-specific nuclease protection assay sandwich hybridization (NPA-SH) probe that targets the large subunit of ribosomal RNA (LSU rRNA). We tested probe specificity and sensitivity with five other dinoflagellates that also cause red tides. Our assay detected H. triquetra at a concentration of 1.5×10⁴ cells/mL, more sensitive than required for a red-tide guidance warning by the Korea Ministry of Oceans and Fisheries in 2015 (3.0×10⁴ cells/mL). We also used the NPA-SH assay to monitor H. triquetra in the Tongyeong region of the southern sea area of Korea during 2014. This method could detect H. triquetra cells within 3 h. Our assay is useful for monitoring H. triquetra under field conditions.     

东海原甲藻抗体的制备及酶联免疫检测方法的建立

通过制备针对东海原甲藻细胞破碎物的多克隆和单克隆抗体,建立了基于双抗体酶联免疫分析定量检测东海原甲藻(Prorocentrum donghaiense)的检测方法。利用该方法对单一藻种、混合藻种和现场样品中的东海原甲藻进行检测的结果与镜检结果相一致,最低检测限度为1×103 cells/m L。该方法的建立对中国近海赤潮暴发的实时监控具有重要意义。     

Optical detection of Prorocentrum donghaiense blooms based on multispectral reflectance

Prorocentrum donghaiense is one of the most common red tide causative dinoflagellates in the Changjiang （Yangtze） River Estuary and the adjacent area of the East China Sea. It causes large-scale blooms in late spring and early summer that lead to widespread ecologic and economic damage. A means for distinguish- ing dinoflagellate blooms from diatom （Skeletonema costatum） blooms is desired. On the basis of measure- ments of remote sensing refectance [Rrs（λ）] and inherent optical parameters, the potential of using a mul- tispectral approach is assessed for discriminating the algal blooms due to P. donghaiense from those due to S. costatum. The behavior of two reflectance ratios [R1 = Rrs（560）/Rrs（532） and Re = Rrs（708）/Rrs（665）], suggests that differentiation of P. donghaiense blooms from diatom bloom types is possible from the current band setup of ocean color sensors. It is found that there are two reflectance ratio regimes that indicate a bloom is dominated by P. donghaiense; （1） R1 〉 1.55 and R2 〈 1.0 or （2） R1 〉 1.75 and R2 ≥ 1.0. Various sensitivity analyses are conducted to investigate the effects of the variation in varying levels of chlorophyll concentration and colored dissolved organic matter （CDOM） as well as changes in the backscattering ratio （bbp/bp） on the efficacy of this muitispectral approach. Results indicate that the intensity and inherent op- tical properties of the algal species explain much of the behavior of the two ratios. Although backscattering influences the amplitude of Rrs（λ）, especially in the 530 and 560 nm bands, the discrimination between P. donghaiense and diatoms is not significantly affected by the variation of bbp/bp. Since aCDOM （440） in coastal areas of the ECS is typically lower than 1.0 m-1 in most situations, the presence of CDOM does not interfere with this discrimination, even as SCDOM varies from 0.01 to 0.026 nm-1. Despite all of these effects, the dis- crimination of P. donghaiense blooms from diatom blooms based on multispectral measurements of Rrs（λ） is feasible.     

海洋原甲藻与三角褐指藻混合培养条件下的种群生长与氮磷营养盐变化

以三角褐指藻(Phaeodactylum tricornutum Bohl)和海洋原甲藻(Prorocentrum mi-cans)为实验种研究了硅藻与甲藻在混合培养的环境中其各自的种群密度变化以及相应水体中氮磷营养盐变化。结果显示,在单独培养或与海洋原甲藻共同培养的条件下,三角褐指藻均表现出明显的营养盐吸收优势,种群生长迅速。相对而言,海洋原甲藻对营养盐的吸收速率明显较低,而且在与三角褐指藻共同培养的水体中一直处于竞争劣势;但有海洋原甲藻存在的水体中,三角褐指藻较单独培养时具有更高的生长速率和高的生物量,海洋原甲藻的存在可能促进三角褐指藻的生长。     

光照和硝酸盐浓度对东海原甲藻和三角褐指藻释放挥发性卤代烃的影响

海洋中产生的挥发性卤代烃(Volatile Halocarbons,VHCs)是氯、溴和碘进入大气的重要载体。海洋藻类能够产生损耗大气中臭氧的VHCs,尤其是海洋微藻已被证明是大气中一些VHCs的主要贡献者。环境因素对海洋微藻产生VHCs的影响研究较少,本文主要研究了光照和硝酸盐浓度对微藻释放VHCs的影响。将海洋微藻东海原甲藻(Prorocentrum donghaiense)和三角褐指藻(Phaeodactylum tricornutum)置于密封的玻璃容器中,并在不同光照条件(20 μmol/(m²·s)、70 μmol/(m²·s)和140 μmol/(m²·s))及不同硝酸盐浓度(1 mg/L、5 mg/L、10 mg/L和50 mg/L)下进行无菌单种培养,分析碘甲烷(CH₃I)、二溴甲烷(CH₂Br₂)、一氯二溴甲烷(CHBr₂Cl)和三氯乙烯(C₂HCl₃)4种VHCs的生产。采用吹扫−捕集气相色谱技术对其中的VHCs进行提取和分析。结果表明,光照强度和硝酸盐浓度会影响两种微藻对VHCs的释放,但是对不同VHCs的影响效果不同,其中CH₃I的释放受光照强度和硝酸盐浓度变化的影响比较显著。一定范围内,光照强度越大,两种微藻对CH₃I的释放量越大。适当的硝酸盐浓度(> 5 mg/L)在一定程度上促进了两种微藻对CH₃I的释放。     

基于现场调查和MODIS卫星遥感下东海原甲藻主导的混合甲藻赤潮生消规律研究

浙江省南部沿海地区是赤潮灾害事件频发的海区之一.本研究通过现场调查以及利用搭载在极地轨道环境遥感卫星Aqua上的中等分辨率成像光谱仪(Moderate Resolution Imaging Spectro Radiometer,MODIS)传感器数据,研究了2016年5月浙江南部洞头和南麂列岛附近海域东海原甲藻(Pro...     

Identification of Provocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified,cloned and sequenced,and these sequence data were deposited in the GenBank.Eight oligonucleotide probes(DNA probes)were designed based on the sequence analysis.The probes were employed to detect and identify P.minimum and T. pulchella in unialgal and mixed algal samples with a fuorescence in situ hybridization method using flow cytometry.Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences,and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe.These DNA probes and the hybridization protocol we developed were specific and effective for P.minimum and T. pulchella,without any specific binding to other algal species.The hyrbridization efficiency of difierent probes specific to P.minimum was in the order:PMl8S02＞PM28S02＞PM28S01＞PM18S01,and that of the probes specific to T. pulchella was TP18S02＞TP28S01＞TP28S02＞TP18S01.The djfferent hybridization efficiency of the DNA probes could also be shown in the fuorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry.The DNA probes PM18S02,PM28S02,TPl8S02 and TP28S01,and the protocol,were also useful for the detection of algae in natural samples.