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PCR检测法鉴定微囊藻及其产毒特性
潘卉
Subtype硕士
Thesis Advisor宋立荣
2000
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline水生生物学
Keyword全细胞pcr 微囊藻 水华蓝藻 微囊藻毒素 基因
Abstract蓝藻水华及其产毒引起的环境问题,已经并将在相当长的时期内,成为我国众多湖泊、水库、池塘面临的首要问题。本文旨在以PCR方法为基础,发展有效而实用的水华蓝藻种属的鉴定方法和产毒特性的早期检测技术,为监测、控制蓝藻水华以及制定水质的安全评估值打基础。本文的主要结果包括以下几个方面:一、用NEST-PCR法扩增16S rRNA中微囊藻属特征序列,结果表明只有微囊藻属藻株才能得到所需扩增片段,确认了本文所检测的微囊藻株。二、RAPD-PCR法通过建立微囊藻属内不同藻株的DNA指纹图谱,区分开微囊藻属内各藻株。作为对照的1株鱼腥藻与各微囊藻藻株的遗传距离大于微囊藻藻株之间的遗传距离,证明此方法结果是可靠的。三、通过PCR引物对MTF/P证实:产毒及非产毒微囊藻中普遍存在着多肽合成酶成因,但不是其他所有蓝藻中都有此基因。四。分别用几种常用的毒素检测方法,即HPLC、ELISA、BIOASSAY确定各藻株用水样的毒性。五、两对引物对TOX1P/1F和TOX2P/2F用于特异地扩增微囊藻毒素合成酶基因Mcy B片段。PCR结果显示,只在产微囊藻毒素的微囊藻中得到所需扩磁片段,不产毒的微囊藻藻株没有扩增片段。PCR法鉴别出的产毒株和非产毒株与其他三种检测方法的检测结果是吻合的。本次检测所有的微囊藻藻株数量较多,从统计学角度排除了产毒特性和基因背景之间相关的偶然性。因此,此方法可用为简单有效区分微囊藻产毒及非产毒株的新方法。六、在此基础上发展的全细胞PCR成功地免去提取藻类DNA的麻烦,用实验室培养物的完整藻,冻存的干藻粉甚至含有不止一种蓝藻的自然一水样做PCR,同样获得较好的检测效果。NEST-PCR和微囊藻毒素合成酶基因特性PCR所用引物都能用于全细胞PCR检测,检测结果与常规PCR反应一致。本方法检测下限达到2000cells/ml以下。以上结果说明,全细胞PCR方法用于检测或监测微囊藻及其产毒特性,简单安全,省时省力,特异性强,检测下限低,对材料、仪器和操作的要求不高且成本低廉,还可同时检测大批量样品,此方法符合鉴定及早期检测、监控微囊藻水华、产毒微囊藻的要求。
Other AbstractThe objective of this work is to develop DNA-based methods for identification of Microcystis strains and for discrimination of toxic and non-toxic strains among Microcystis and other related blooming-forming cyanobacteria. Our studies include three parts: 1. Identification of genus Microcystis via NEST-PCR; 2. Differentiation of seven Microcystis strains by RAPD; and 3. Establishment of whole cells PCR in discriminating toxic and non-toxic cyanobacteria. In order to identify Microcystis strains, Microcystis-specific PCR primers was designed according to the regions V6, V7, V8 of 16S rRNA. As illustrated by the NEST-PCR, all the Microcystis strains have the target amplification fragments, while the Aphanizomenon flos-aquae, Anabaena flos-aquae, Oscillatoria planctonica, Anabaena sp., Synechococcous elenpata show no aimed product. our results confirm the accuracy of the strains to be tested in the further research. In the second part of our study, randomly amplified polymorphic DNA PCR was used to get DNA profiles of 7 Microcystis strains. The genetic distance between Anabaena and Microcystis is over 0.68; whereas the values range from 0.073 to 0.507 among 7 strains of Microcystis. The results suggest that randomly amplified polymorphic DNA PCR is relatively reliable to infer genetic relation and evolution of Microcystis and is proved to be an alternative and complementary approach to the traditional methods to differentiate the Microcystis strains. The third part of our study aimed to answer: Do cyanobacteria in general possess peptide synthetase genes or is the presence of these genes limited to a few species? Is the occurrence of the mcy B gene limited to toxic Microcystis aeruginosa? meanwhile, we also aimed to develop a simple and sensitive protocol being table to set apart toxic and non-toxic strains in cyanobacteria, particularly in microcystis. Amplified with degenerate primers MTF/MTR, all the Microcystis strains and Aphanizomenon flos-aquae 44-1, Anabaena flos-aquae 1444 contain peptide synthetase genes, even when they are non-toxic. Oscillatoria planctonica 708 gave negative signal. Based on the evidence that Mcy B is closely related to microcystin biosynthesis, we have surveyed the distribution of the Mcy B gene in different cyanobacterial species. For this purpose, two primer pairs (Tox1P/1M; Tox2P/2M) were designed and specifically used to detect the mcy B gene. The primers were able to amplify fragments of the mcy B gene from DNA of all known microcystin producers; all the non-toxic strains being tested did not yield aimed PCR product. The result thus clearly demonstrated that it is quite possible to distinguish microcystin with non-microcystin peptide synthetases gene in the Microcystis using DNA techniques. To ensure the accuracy of the PCR, we checked the toxicity of all strain by HPLC, Mouse Bioassay and ELISA. All these methods test and verify the results of PCR. Furthermore, our study has created the 'Whole Cells PCR' protocol which has been proved to be a simpler and more sensitive method in discriminating the microcystin-producing and non-microcystin-producing Microcystis strains. The potential application of this protocol in monitoring and evaluating the natural waters samples has been emphasized and discussed in the light of our findings.
Pages72
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12564
Collection学位论文
Recommended Citation
GB/T 7714
潘卉. PCR检测法鉴定微囊藻及其产毒特性[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,2000.
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