应用荧光原位杂交方法检测中国沿海塔玛/链状亚历山大藻复合种(亚洲温带基因型)

近年来,有害赤潮于中国沿海频繁发生,对沿海居民身体健康、水产养殖和自然生态形成了潜在的威胁.由于部分有毒赤潮藻在很低的密度下就有可能导致严重的危害,但传统的采样和分析方法无法对这类有毒赤潮进行有效的检测,因而急需发展准确、高效的检测新方法.根据对中国沿海分离的塔玛/链状亚历山大藻(亚洲温带基因型)核糖体大亚基DNA(LSU rDNA)序列信息的分析,设计了两条特异性的荧光标记探针,并建立了针对中国沿海塔玛/链状亚历山大藻复合种(亚洲温带基因型)的荧光原位杂交检测方法室内模拟实验显示两条探针都能够特异性地标记中国沿海塔玛/链状亚历山大藻(亚洲温带基因型),但标记效果有一定差异,探针SPEC-PROBE2标记效果远好于探针SPEC-PROBE1.经过标记的藻细胞可以通过荧光显微镜和流式细胞仪区分.中国沿海塔玛/链状亚历山大藻荧光原位杂交检测方法的建立将有助于提高海水样品中亚历山大藻监测的准确性和工作效率.     

中国东南沿海四株亚历山大藻的系统发育研究

通过核糖体基因大亚基(LSU rDNA)D1-D2区以及ITS序列分析,研究了采自于中国东南沿海的4株亚历山大藻(Alexandrium Halim)的系统发育.结果显示,本地的亚历山大藻与来自日本和韩国的藻株亲缘关系很近;而且通过构建系统发育树,发现本地亚历山大藻与东亚藻株聚成一个大族.与东亚藻株在rDNA序列上的高相似性也说明本地株亚历山大藻属于温带亚洲基因型,而从欧美传入的机会较小.结果同时显示,塔玛亚历山大藻(A. tamarense)与链状亚历山大藻(A. catenella)LUS和ITS序列相近,可能在遗传学上是同一种类.     

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.     

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.     

羟基自由基对塔玛亚历山大藻DNA破坏研究

利用羟基自由基(·OH)压载水处理系统,采用大气压强电场放电技术制取·OH溶液对塔玛亚历山大藻(Alexandrlum tamarense)进行处理。通过普通光学显微镜,荧光显微镜和电子显微镜对·OH处理前后的塔玛亚历山大藻的细胞结构进行观测。结果表明,·OH能有效破坏藻细胞,从而造成藻类死亡。利用随机扩增多态性DNA(random amplification polymorphic DNA,RAPD)和实时定量PCR(RT-PCR)相结合的技术检测·OH对DNA链的破坏作用。共得到了3条有显著差异的扩增产物。这3条扩增产物经测序,并通过NCBI(national center of biotechnology information)的比对分析,最终得到1条可用RT-PCR检测·OH对DNA破坏作用的基因序列。以上的结果表明,·OH压载水处理系统能有效去除塔玛亚历山大藻,并对其DNA造成破坏。     

大型海藻与赤潮微藻以及赤潮微藻之间的相互作用研究

研究了2种大型海藻石莼(Ulva pertusa)和江蓠(Gracilaria lemaneiformis)对2种赤潮微藻:东海原甲藻(Prorocentrumdonghaiense)和塔玛亚历山大藻(Alexandum tamarense)生长的影响以及2种微藻之间的相互作用,结果发现:①在大藻(石莼或江蓠)-微藻(东海原甲藻或塔玛亚历山大藻)的共培养体系中,石莼和江蓠均能明显影响与其共培养的微藻的生长,石莼对微藻生长的影响强于江蓠的作用.②在东海原甲藻-塔玛亚历山大藻的双藻培养体系中,东海原甲藻的生长受到明显的抑制作用,最终被完全灭杀;体系中塔玛亚历山大藻的生长未受到明显的影响.另外,塔玛亚历山大藻的培养液滤液能明显抑制东海原甲藻的生长,但东海原甲藻滤液对塔玛亚历山大藻的生长几乎没有影响.实验室条件下模拟二者相互作用的结果显示,塔玛亚历山大藻对东海原甲藻的抑制作用是其被后者抑制作用的17倍左右.③在大藻(石莼或江蓠)-东海原甲藻-塔玛亚历山大藻的多藻培养体系中,东海原甲藻和塔玛亚历山大藻的生长变化与它们在共培养体系中的变化非常类似.半数致死时间(LT50)法的检测结果显示:多藻培养体系对东海原甲藻的联合作用是协同作用,而对塔玛亚历山大藻的作用是相加作用.     

溶藻细菌BS03分离、鉴定及其对塔玛亚历山大藻生长的影响

从福建漳江口红树林区筛选出一株对产贝毒赤潮原因藻——塔玛亚历山大藻(Alexandrium tamatense)具有强溶藻能力的细菌,命名为BS03,通过生理生化及16S rDNA序列分析对该菌株进行鉴定,并探讨了菌株BS03对塔玛亚历山大藻的溶藻特性和溶藻代谢产物的初步性质.结果发现,菌株BS03属于微泡菌属(Microbulbifer sp.)相似性达99％;对塔玛亚历山大藻的杀藻效果具有一定浓度效应,在一定浓度范围内处理浓度越高,溶藻效果越好;菌株BS03对处于不同生长时期的塔玛亚历山大藻都表现出较好的杀藻效果,其中对处于延滞期的塔玛亚历山大藻表现出最佳的杀藻效果,处理96 h后,抑藻率达98.17％;不同生长期菌株对塔玛亚历山大藻溶藻作用无明显差异;菌株BS03通过间接作用方式溶藻,所分泌的胞外活性物质的分子量小于1 kDa,耐酸碱、具热稳定性,推测为非蛋白质、非核酸和非多糖类物质.     

CO_2加富对塔玛亚历山大藻和小新月菱形藻种群竞争的影响

通过共培养的方法,研究了塔玛亚历山大藻(Alexandrium tamarense)和小新月菱形藻(Nitzschia closteriumEhr)种群竞争关系对CO2加富的响应变化。培养体系中CO2的浓度分别保持在360μL/L(未加富)和5 000μL/L(CO2加富)。结果表明:(1)不同的初始接种密度对2种藻种群竞争有明显的影响。当塔玛亚历山大藻(A)和小新月菱形藻(N)的初始接种密度比为A∶N=1∶4时,小新月菱形藻在竞争中占有极为明显的优势;当接种比例为A∶N=1∶1,小新月菱形藻占有一定的竞争优势;当接种比例为A∶N=4∶1时,塔玛亚历山大藻在竞争中占有明显优势。(2)CO2加富可改变2种藻种群竞争的关系,使塔玛亚历山大藻竞争能力大大提高,小新月菱形藻种群竞争能力降低,从而导致接种比例A:N=1:4时,塔玛亚历山大藻的竞争劣势不再明显;而接种比例为A:N=1:1和4:1时,塔玛亚历山大藻成为竞争中的优势种。     

蒙古裸腹溞对有毒赤潮生物塔玛亚历山大藻的摄食研究

在实验室条件下研究了枝角类蒙古裸腹溞(Moina mongolica)对有毒赤潮生物塔玛亚历山大藻的摄食。结果表明,单一饵料试验中,蒙古裸腹溞摄食率在一定范围内随塔玛亚历山大藻细胞数的增加而增大,在塔玛亚历山大藻和亚心形扁藻组成的混合饵料试验中,当前者细胞数为低值时,摄食率不随后者细胞数的变化而变化,而当前者的细胞数高于1920/mL时,摄食率随亚心形扁藻细胞数的增大而升高。     

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。     

Demonstration of spontaneously dividing male fetal cells in maternal blood by negative magnetic cell sorting and fish

We investigated a case of massive feto-maternal bleeding by using negative magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH). A 37-year-old pregnant woman had an uncomplicated amniocentesis for advanced maternal age at 16 weeks' gestation. The fetal karyotype was 46, XY. At 19 weeks' gestation, she had a minor car accident and slight vaginal bleeding. A subsequent Kleihauer-Betke test showed a 140 ml feto-maternal haemorrhage. Serial sonographic examinations indicated a normal fetus and placenta. We performed FISH analysis on maternal peripheral blood at 25 weeks. Anti-CD45 and MACS were used to deplete maternal leucocytes, enriching the proportion of fetal nucleated erythrocytes present. The isolated cells were analysed by using dual-colour FISH with X and Y specific probes. Approximately 65 800 nucleated cells were obtained after MACS depletion. A total of 234 cells were analysed by FISH. The results revealed that 70 of the nucleated cells (30 per cent) were male with one X and one Y signal. Among these cells, six male metaphases were observed in spontaneously dividing cells.     

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.     

Fully automated FISH examination of amniotic fluid cells

Objective Fluorescence in situ hybridization (FISH) analysis has become a valuable adjunct in cytogenetics, providing a rapid screen for common chromosome abnormalities that is particularly helpful in prenatal diagnosis. FISH analysis using standard microscopy is expensive and labor intensive, requiring both a high skill level and subjective signal interpretation. A reliable fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. Methods The efficacy of an automated system was compared to standard manual FISH analysis. Two sets of slides were generated from each of 152 amniotic fluid samples. Following hybridization with a standard panel of five chromosome FISH probes, one set of slides was evaluated using manual microscopy. The other set was evaluated using an automated microscopy system. Results A diagnostic outcome was obtained for all 152 samples using manual microscopy and for 146 of 152 (96%) samples using automated microscopy. Three cases of aneuploidy were detected. For those samples for which a diagnostic outcome was determined by both manual and automated microscopy, 100% concordance was observed. All FISH analysis results were confirmed by karyotype. Conclusion These data suggest that an automated microscopy system is capable of providing accurate and rapid enumeration of FISH signals in amniocytes. Copyright © 2007 John Wiley & Sons, Ltd.     

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.     

AZFc deletion detected in a newborn with prenatally diagnosed Yq deletion

A case of prenatally diagnosed Yq deletion is described. Fluorescence in situ hybridisation (FISH) was used to identify the abnormal chromosome and to exclude mosaicism. Based on the cytogenetic result and the ultrasound investigation the pregnancy was continued. A newborn with normal male genitalia was delivered. Microdeletion analysis of the Yq showed the absence of the AZFc region. This type of deletion has been described as being associated with azoospermia or oligozoospermia with a progressive decrease of sperm number over time. Long-term andrological follow-up of the newborn will be necessary with eventual cryoconservation of sperm at early adulthood. The present report proposes that AZF analysis combined with FISH has an important role in accurate genetic counselling in sex chromosome anomalies. Copyright © 2001 John Wiley & Sons, Ltd.     

基于成组生物毒性测试的PM2.5毒性

为了准确评估PM_(2.5)的综合生物毒性,采用发光细菌急性毒性、斑马鱼胚胎急性毒性和体外人肺腺癌细胞(A549)毒性试验对常州市冬季雾-霾天、冬季正常天和夏季这3个时间段的PM_(2.5)进行生物毒性评价,并结合大气理化指标进行分析.结果表明,3个时间段的PM_(2.5)均表现出急性毒性或发育毒性.冬季雾-霾天、冬季正常天和夏季PM_(2.5)对发光细菌的毒性单位(toxicity unit,TU)值分别为1. 74(有毒)、1. 19(有毒)和0. 92(微毒);对斑马鱼胚胎的TU值从大到小排列为:冬季正常天(TU=1. 14,有毒)、冬季雾-霾天(TU=0. 79,微毒)和夏季(TU=0,无毒);对A549的TU值为:冬季雾-霾天(TU=0. 61,微毒)夏季(TU=0. 38,微毒)冬季正常天(TU=0. 31,微毒).在发育毒性方面,除夏季PM_(2.5)样品,其余2个时间段的PM_(2.5)均对斑马鱼胚胎发育有影响,主要表现出心包囊肿、脊椎弯曲和尾部畸形.平均毒性(average toxicity,Av Tx)、毒性指数(toxic print,Tx Pr)和最敏感测试(most sensitive test,MST)综合毒性评价方法表明冬季雾-霾天和冬季正常天PM_(2.5)均表现出有毒,夏季则表现出微毒,以冬季雾-霾天PM_(2.5)样品的综合毒性最高.此外,3种测试生物对PM_(2.5)污染物的敏感度排序为发光细菌斑马鱼胚胎 A549.理化指标和生物毒性的相关性分析结果表明,PM_(2.5)中所含的污染物对生物毒性效应产生影响,可为PM_(2.5)生物毒性综合评价和人体健康风险评估提供依据.     

Low or absent unconjugated estriol in pregnancy: an indicator for steroid sulfatase deficiency detectable by fluorescence in situ hybridization and biochemical analysis

It has been previously reported that a low or absent maternal serum unconjugated estriol (uE3) level is associated with placental steroid sulfatase (STS) deficiency. Here we report a correlation between patients who present with a very low or absent maternal serum uE3 and a deletion of the STS gene as assessed by fluorescence in situ hybridization (FISH). We studied nine prenatal cases that presented to the clinical laboratory with an abnormal triple screen, specifically low or absent maternal serum uE3 and a 46,XY karyotype. FISH analysis showed complete deletion of a probe containing the STS gene in six cases and one case had a partial deletion (reduced but not absent signal). The remaining two cases were not deleted for the STS probe. All mothers tested whose fetus showed a deletion were shown to be STS deletion carriers using FISH. Biochemical analysis was performed on 7/9 prenatal specimens. All fetuses deleted for the STS probe were also found to be deficient for STS by biochemical analysis of cultured amniotic fluid (5/5). Of the two fetuses not deleted for the STS probe, one was deficient for STS activity, while the other had a normal result. The abnormal result of enzyme deficiency by biochemical analysis in a non-deletion case likely represents a mutation in the STS gene, not detectable by this FISH assay. Postnatal FISH confirmation of the STS deletion was performed in 1/7 cases. Clinical follow-up was available for 4/9 cases following birth. Copyright © 2002 John Wiley & Sons, Ltd.     

北部湾卡盾藻(Chattonella spp.)种类鉴定

2018年9月在北部湾海域分离得到3株卡盾藻,采用光学显微镜对其进行形态学初步鉴定,并通过核糖体大亚基LSU rDNA和ITS基因进行系统进化分析。结果表明,卡盾藻北部湾株符合海洋卡盾藻形态学典型特征:藻体黄褐色,细胞呈纺锤形,无黏液泡,尾部具尖端,细胞平均长（57.8±9.1）μm。系统发育树上北部湾卡盾藻分别以96%（LSU rDNA）和99%（ITS）的自展支持率与不同来源的海洋卡盾藻、古老卡盾藻、卵圆卡盾藻、小型卡盾藻聚在同一大分支上。通过遗传距离分析发现,北部湾3株卡盾藻与不同来源的海洋卡盾藻、古老卡盾藻、卵圆卡盾藻、小型卡盾藻亲缘关系较近,LSU和ITS遗传距离分别为0.0000~0.0016和0.0000~0.0235;而与盐生卡盾藻亲缘关系较远,遗传距离分别为0.0415~0.0490和0.0733~0.0750。因此,综合形态学和分子生物学结果可以确定北部湾3株卡盾藻为海洋卡盾藻/海洋卡盾藻的变种。研究结果可为我国卡盾藻藻华藻种库的构建和卡盾藻北部湾株生理生态学的研究提供基础数据。