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研究生：	尤麗塔 Marbun, Yovita Ramos
論文名稱：	發展產柱孢藻毒素藍綠菌快速監測技術之研究 Development of Rapid On-site Monitoring Technique for Toxigenic Cylindrospermopsis Cyanobacteria
指導教授：	林財富 Lin, Tsair-Fuh
學位類別：	碩士 Master
系所名稱：	工學院 - 環境工程學系 Department of Environmental Engineering
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	93
外文關鍵詞：	Cylindrospermopsis raciborskii, cylindrospermopsin, rapid on-site monitoring, quantitative polymerase chain reaction (qPCR), drinking water reservoirs
相關次數：	點閱：122 下載：1
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The cyanobacterium Cylindrospermopsis raciborskii widely distributes across the temperate, subtropical, and tropical world and becomes a concern in water quality perspective due to its toxin cylindrospermopsin in drinking and recreational water. Therefore, an effective and efficient on site monitoring and management strategies of C. raciborskii is needed to minimize the harmful effects of cylindrospermopsin.
Rapid detection method for on-site monitoring of toxigenic C. raciborskii, combining portable quantitative polymerase chain reaction (qPCR) and DNA extraction by microwave disruption, developed and tested in Cheng Kung Lake, National Cheng Kung University and drinking water reservoirs. Based on rpoC1 and polyketide synthase (pks) copy number, qPCR is able to quantify the cell number of total C. raciborskii and cylindrospermopsin-producer simutaneously. Furthermore, the impact of the water matrix on qPCR analysis was also investigated. It describes that there are no difference between environmental samples and JM medium in the Ct values.
From Cheng Kung Lake and Kinmen Samples, the result of total C. raciborskii agreed with counting cells result by microscope. While the result of toxigenic cells in Kinmen samples consistent with the result of cylindrospermopsin concentration. The results indicate that qPCR employed here was able to quantify C. raciborskii and cylindrospermopsin producers within 2 hours after sampling with detection limit at 5000 cells/ml. This accurate predictive information can be used to guide remedial measures of toxic blooms in advance.

TABLE OF CONTENTS


ABSTRACT                                        			i
ACKNOWLEDGEMENTS                                        		ii
TABLE OF CONTENTS                                        		iv
LIST OF FIGURES                                        		vii
LIST OF TABLES                                        		x

CHAPTER	I		INTRODUCTION  		1
1.1	Background  		1
1.2	Objectives  		2
1.3	Scope of work  		2
1.4	Methodology  		3
1.5	Thesis overview  		4

CHAPTER	II	LITERATURE REVIEW  		5
		2.1	Cyanobacteria  		5
		2.2	Cylindrospermopsis raciborskii  		7
		2.3	Cylindrospermopsin  		8
		2.3.1	Biosynthesis pathway 		10
		2.4	Detection method for Cylindrospermopsis raciborskii and cylindrospermopsin  		11
		2.4.1	Detection method for Cylindrospermopsis raciborskii  		11
		2.4.2	Detection method for cylindrospermopsin  		12
		2.5	Molecular biology techniques  		15
		2.5.1	Molecular cloning  		16
		2.5.2	Polymerase Chain Reaction (PCR) and Gel Electrophoresis  		17
		2.5.3	Real-time Polymerase Chain Reaction (qPCR)  		20
		2.5.4	The application of molecular biology and genomics to 	
				environmental microbiology  		22
		2.5.5	Rapid testing for toxigenic Cylindrospermopsis  		23
		2.6	Rapid preparation of cyanobacterial DNA  		23

CHAPTER	III	MATERIALS AND METHODS  		26
		3.1	  Cylindrospermopsis raciborskii strain and culturing  		26
		3.2	  Cell counting by Microscope  		26
		3.3	  DNA extraction and cell disruption  		26
		3.4	  Oligonucleotide primers  		27
		3.5	  PCR (Polymerase Chain Reaction)  		29
		3.6	  Gene cloning  		29
		3.7	  qPCR  		30
		3.8	  Enzyme-linked immunosorbent assay (ELISA)  		30
		3.9	  On-site monitoring  		31

CHAPTER	IV	RESULTS AND DISCUSSIONS  		37
		4.1	  Development of qPCR method for C. raciborskii  		37
		4.1.1	  Primer selection for C. raciborskii and cylindrospermopsin specific sequences  		37
		4.1.2	  PCR condition optimization  		38
		4.1.3	  Optimization of qPCR condition  		42
		4.2	  Development of rapid DNA extraction method (Microwave Cell Disruption) for onsite monitoring  		49
		4.2.1	  Estimation of C. raciborskii cell concentration  		49
		4.2.2	  Investigation of water matrix impact  		51
		4.2.3	  Comparison between qPCR and microscopic cell count  		53
		4.2.4	  Method detection limit  		55
		4.3	  On-site monitoring application  		57
		4.3.1	  Cheng Kung Lake samples  		57
		4.3.2	  Kinmen samples  		58
		4.3.3	  Limitation of the methods  		61

CHAPTER	V	CONCLUSIONS  		63
		5.1	Conclusions  		63
		5.2	Suggestions  		64

REFERENCES  		65

APPENDIX A 	Detail of Materials and Methods		69
		A.1	  JM medium  		69
		A.2	  Gene cloning experiment  		70
		A.3	  Statistical Analysis (Two Way ANOVA)  		74
		A.4	  Method detection limit  		75
		A.5	  Detection specificity  		75
APPENDIX B 	Development of Taqman Probe Method  		76
APPENDIX C 	Result of On-site monitoring		77
		C.1	  Sample from Cheng Kung Lake  		77
		C.2	  Sample from Bao Shan 2 Reservoir and Li Yu Lake  		79
		C.3	  Sample from Kinmen  		80
APPENDIX D 	Growth curve experiments		82
APPENDIX E 	Figure of equipments		89
 

                                    

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