针对微囊藻毒素合成酶基因mcyA探索一种适用于自然水样中微囊藻毒素产毒潜能检测的TaqMan实时荧光定量PCR方法。该方法从铜绿微囊藻FACHB-1279中的mcyA基因中扩增出目的基因片段,用TA克隆方法将基因片段插入Allel TA vector(ABP-CE-TAVE...针对微囊藻毒素合成酶基因mcyA探索一种适用于自然水样中微囊藻毒素产毒潜能检测的TaqMan实时荧光定量PCR方法。该方法从铜绿微囊藻FACHB-1279中的mcyA基因中扩增出目的基因片段,用TA克隆方法将基因片段插入Allel TA vector(ABP-CE-TAVEC020)中来制备质粒标准品。通过不同连续稀释倍数的质粒标准品成功构建了mcyA基因的标准曲线,该法构建的标准曲线相关系数高达0.998 3,满足实时荧光定量RT-PCR的要求。该方法检测所需水样少(仅200μL),具有水样基因组提取操作简便、快速、线性范围广、灵敏度高等优点,能应用于水库、湖泊等水源中具有产毒性能的微囊藻的快速定量检测。展开更多
Eutrophication or the process of nutrient enrichment of stagnant waters due to excessive use of fertilizer is becoming a critical issue worldwide. Lake Gregory, an artificial lake situated in Nuwara Eliya, Sri Lanka w...Eutrophication or the process of nutrient enrichment of stagnant waters due to excessive use of fertilizer is becoming a critical issue worldwide. Lake Gregory, an artificial lake situated in Nuwara Eliya, Sri Lanka was once a very attractive landscape feature and recreational area attracting a large number of visitors. Rapid urbanization in surrounding areas and the consequent intensification of agricultural and industrial activities led to eutrophication and siltation in the lake. Present study was conducted to detect cyanobacterial diversity and their ability to produce hepatotoxic microcystins using polymerase chain reaction (PCR)-based techniques. Twenty five water samples (surface and bottom) were collected from the lake and total nitrogen and total carbon were estimated. Cyanobacterial cultures were grown in appropriate media and microscopic observations were used to determine the morphological diversity of cyanobacteria isolated from different sites. Genomic DNA was isolated and purified from cyanobacteria using Boom's method. DNA samples were analyzed by PCR with oligonucleotide primers for 16S rRNA gene and mcyA gene of the operon that encodes a microcystin synthetase. The 16S rRNA gene sequences revealed the presences of cyanobacteria belong to Synechococcus sp., Microcystis aeruginosa, Calothrix sp., Leptolyngbya sp., Limnothrix sp., order Oscillatoriales and order Chroococcales. The sequences obtained from this study were deposited in the database under the accession numbers (GenBank: GU368104-GU368116). PCR amplification of mcyA primers indicated the potential for toxin formation of isolated M. aeruginosa from Lake Gregory. This preliminary study shows that the Lake Gregory is under the potential risk of cyanobacterial toxicity. Clearly more work is needed to extend this finding and clarify if other cyanobacterial isolates have genetic potential to produce microcystin since this lake is utilized for recreational activities.展开更多
A water bloom sample collected from Lake Dishui in Shanghai was characterized. The morphological identification showed that Micorcystis wesenbergii and Micorcystis smithii were the main component of the bloom. Five st...A water bloom sample collected from Lake Dishui in Shanghai was characterized. The morphological identification showed that Micorcystis wesenbergii and Micorcystis smithii were the main component of the bloom. Five strains of M. smithii were successfully isolated. Their 16S rRNA gene sequences based phylogenetic tree showed that the five strains of M. smithii intermixed with strains of other morphospecies in Microcystis. A fragment of mcy gene encoding for microcystin synthetase was detected in one of the five M. smithii strains （CHAB 2183）, indicating its potential of microcystin production. High performance liquid chromatography analysis confirmed M. smithii CHAB 2183 to produce microcystin-RR as 1550 μg per gram dry weight cells. The present investigation, for the first time, reported the isolated strains of M. smithii and microcystin production from M. smithii.展开更多
文摘针对微囊藻毒素合成酶基因mcyA探索一种适用于自然水样中微囊藻毒素产毒潜能检测的TaqMan实时荧光定量PCR方法。该方法从铜绿微囊藻FACHB-1279中的mcyA基因中扩增出目的基因片段,用TA克隆方法将基因片段插入Allel TA vector(ABP-CE-TAVEC020)中来制备质粒标准品。通过不同连续稀释倍数的质粒标准品成功构建了mcyA基因的标准曲线,该法构建的标准曲线相关系数高达0.998 3,满足实时荧光定量RT-PCR的要求。该方法检测所需水样少(仅200μL),具有水样基因组提取操作简便、快速、线性范围广、灵敏度高等优点,能应用于水库、湖泊等水源中具有产毒性能的微囊藻的快速定量检测。
文摘Eutrophication or the process of nutrient enrichment of stagnant waters due to excessive use of fertilizer is becoming a critical issue worldwide. Lake Gregory, an artificial lake situated in Nuwara Eliya, Sri Lanka was once a very attractive landscape feature and recreational area attracting a large number of visitors. Rapid urbanization in surrounding areas and the consequent intensification of agricultural and industrial activities led to eutrophication and siltation in the lake. Present study was conducted to detect cyanobacterial diversity and their ability to produce hepatotoxic microcystins using polymerase chain reaction (PCR)-based techniques. Twenty five water samples (surface and bottom) were collected from the lake and total nitrogen and total carbon were estimated. Cyanobacterial cultures were grown in appropriate media and microscopic observations were used to determine the morphological diversity of cyanobacteria isolated from different sites. Genomic DNA was isolated and purified from cyanobacteria using Boom's method. DNA samples were analyzed by PCR with oligonucleotide primers for 16S rRNA gene and mcyA gene of the operon that encodes a microcystin synthetase. The 16S rRNA gene sequences revealed the presences of cyanobacteria belong to Synechococcus sp., Microcystis aeruginosa, Calothrix sp., Leptolyngbya sp., Limnothrix sp., order Oscillatoriales and order Chroococcales. The sequences obtained from this study were deposited in the database under the accession numbers (GenBank: GU368104-GU368116). PCR amplification of mcyA primers indicated the potential for toxin formation of isolated M. aeruginosa from Lake Gregory. This preliminary study shows that the Lake Gregory is under the potential risk of cyanobacterial toxicity. Clearly more work is needed to extend this finding and clarify if other cyanobacterial isolates have genetic potential to produce microcystin since this lake is utilized for recreational activities.
基金supported by the State Key Basic Research and Development Plan of China (No.2008CB418002)the Frontier Research Project of the Chinese Academy of Science (No.082303-1-501)
文摘A water bloom sample collected from Lake Dishui in Shanghai was characterized. The morphological identification showed that Micorcystis wesenbergii and Micorcystis smithii were the main component of the bloom. Five strains of M. smithii were successfully isolated. Their 16S rRNA gene sequences based phylogenetic tree showed that the five strains of M. smithii intermixed with strains of other morphospecies in Microcystis. A fragment of mcy gene encoding for microcystin synthetase was detected in one of the five M. smithii strains （CHAB 2183）, indicating its potential of microcystin production. High performance liquid chromatography analysis confirmed M. smithii CHAB 2183 to produce microcystin-RR as 1550 μg per gram dry weight cells. The present investigation, for the first time, reported the isolated strains of M. smithii and microcystin production from M. smithii.
