Molecular tools for the detection and quantification of toxigenic cyanobacteria

Content: List of Contributors xix About the Editors xxiii About the Book xxvii Preface xxix Acknowledgments xxxi 1 Introduction 1Rainer Kurmayer, Kaarina Sivonen, and Nico Salmaso 1.1 A Brief Historical Overview 1 1.2 The Genetic Basis of Toxin Production 2 1.2.1 Microcystin and Nodularin 2 1.2.2 Cylindrospermopsin 5 1.2.3 Saxitoxin 6 1.2.4 Anatoxin 8 1.3 Application of Molecular Tools 8 1.4 Laboratory Safety Issues 13 1.5 References 14 2 Sampling and Metadata 19Rainer Kurmayer, Guntram Christiansen, Konstantinos Kormas, Wim Vyverman, Elie Verleyen, Vitor Ramos, Vitor Vasconcelos, and Nico Salmaso 2.1 Introduction 19 2.2 Handling of Samples 20 2.3 Sample Contamination 21 2.4 Sampling 21 2.4.1 Quantitative Depth-Integrated and Discrete Sampling 21 2.4.2 Qualitative Plankton Net Sampling 22 2.4.3 Surface (Scum Material) Sampling 22 2.4.4 Benthic (Terrestrial) Cyanobacteria Sampling 22 2.4.5 Food Supplement Sampling 22 2.4.6 Isolation of Single Colonies/Filaments 22 2.5 Subsampling Food Supplement Samples 23 2.6 Sampling of Nucleic Acids 23 2.7 General Conclusions 24 2.8 References 24 SOP 2.1 Sampling and Filtration (DNA) 26Rainer Kurmayer and Konstantinos Kormas SOP 2.1.1 Introduction 26 SOP 2.1.2 Experimental 26 SOP 2.1.3 Procedure 27 SOP 2.1.4 Notes 28 SOP 2.1.5 References 29 SOP 2.2 Sampling of Benthic Cyanobacteria 29Wim Vyverman and Elie Verleyen SOP 2.2.1 Introduction 29 SOP 2.2.2 Experimental 30 SOP 2.2.3 Procedure 30 SOP 2.2.4 Notes 31 SOP 2.2.5 References 31 SOP 2.3 Isolation of Single Cyanobacteria Colonies/Filaments 32 Rainer Kurmayer SOP 2.3.1 Introduction 32 SOP 2.3.2 Experimental 32 SOP 2.3.3 Procedure 33 SOP 2.3.4 Notes 33 SOP 2.3.5 References 33 SOP 2.4 Sampling Food Supplements 34Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos SOP 2.4.1 Introduction 34 SOP 2.4.2 Experimental 35 SOP 2.4.3 Procedure (Fig. 8.3) 35 SOP 2.4.4 Notes 36 SOP 2.4.5 References 36 SOP 2.5 Sampling and Filtration (RNA) 37Rainer Kurmayer and Guntram Christiansen SOP 2.5.1 Introduction 37 SOP 2.5.2 Experimental 37 SOP 2.5.3 Procedure 38 SOP 2.5.4 Notes 38 SOP 2.5.5 References 38 SOP 2.6 Sampling of Abiotic and Biotic Data and Recording Metadata 39Elie Verleyen, Maxime Sweetlove, Dagmar Obbels, and Wim Vyverman SOP 2.6.1 Introduction 39 SOP 2.6.2 Experimental 39 SOP 2.6.3 Type of Metadata and Additional Biotic and Abiotic Data 40 SOP 2.6.4 Notes 41 SOP 2.6.5 References 42 3 Isolation, Purification, and Cultivation of Toxigenic Cyanobacteria 43Sigrid Haande, Iwona Jasser, Muriel Gugger, Camilla H.C. Hagman, Annick Wilmotte, and Andreas Ballot 3.1 Introduction 43 3.2 Methodical Principles for Cyanobacterial Isolation, Purification, and Cultivation 44 3.2.1 Sampling, Identification, and Treatments Prior to the Isolation of Cyanobacteria 44 3.2.2 Traditional Techniques for the Isolation and Purification of Cyanobacteria 45 3.2.3 Culture Media Preparation 47 3.2.4 Cultivation Conditions 48 3.3 General Conclusions 49 3.4 References 49 SOP 3.1 Isolation, Purification, and Clonal Isolate Testing 51Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot SOP 3.1.1 Introduction 51 SOP 3.1.2 Experimental 51 SOP 3.1.3 Procedure 52 SOP 3.1.4 Notes 54 SOP 3.1.5 References 54 SOP 3.2 Isolation of Picocyanobacterial Cells by Flow Cytometer (FCM) Sorting 55Ewa Koz(3)owska and Iwona Jasser SOP 3.2.1 Introduction 55 SOP 3.2.2 Experimental 56 SOP 3.2.3 Procedure 56 SOP 3.2.4 Notes 58 SOP 3.2.5 References 59 SOP 3.3 Axenization 60Muriel Gugger SOP 3.3.1 Introduction 60 SOP 3.3.2 Experimental 60 SOP 3.3.3 Procedure 61 SOP 3.3.4 Notes 63 SOP 3.3.5 References 63 SOP 3.4 Culture Media (Solid and Liquid) 64Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot SOP 3.4.1 Introduction 64 SOP 3.4.2 Experimental 64 SOP 3.4.3 Procedure 65 SOP 3.4.4 Notes 68 SOP 3.4.5 References 68 SOP 3.5 Strain Maintenance (Living Cultures) 69Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot SOP 3.5.1 Introduction 69 SOP 3.5.2 Experimental 69 SOP 3.5.3 Procedure 70 SOP 3.5.4 Notes 72 SOP 3.5.5 References 73 SOP 3.6 Cryopreservation and Recovery 73Muriel Gugger SOP 3.6.1 Introduction 73 SOP 3.6.2 Experimental 74 SOP 3.6.3 Procedure 75 SOP 3.6.4 Notes 78 SOP 3.6.5 References 78 4 Taxonomic Identification of Cyanobacteria by a Polyphasic Approach 79Annick Wilmotte, H. Dail Laughinghouse IV, Camilla Capelli, Rosmarie Rippka, and Nico Salmaso 4.1 Introduction 79 4.2 Nomenclature and Classification of Cyanobacteria 82 4.3 Microscopy 84 4.3.1 Light Microscopy 84 4.3.2 Autofluorescence Microscopy 86 4.4 Molecular Markers: Single Loci 87 4.5 Molecular Markers: Multiple Loci 94 4.5.1 Multilocus Sequence Typing (MLST) and Multilocus Sequence Analysis (MLSA) 94 4.5.2 Genome-Based Extension of MLST and MLSA 96 4.6 Molecular Typing Methods Based on Gel Electrophoresis 96 4.7 Denaturing Gradient Gel Electrophoresis (DGGE) 97 4.8 Taxonomic and Molecular Databases 97 4.9 The Polyphasic Approach 98 4.10 Final Considerations 105 4.11 References 106 SOP 4.1 Taxonomic Identification by Light Microscopy 120Nico Salmaso, Rosmarie Rippka, and Annick Wilmotte SOP 4.1.1 Introduction 120 SOP 4.1.2 Experimental 121 SOP 4.1.3 References 124 SOP 4.2 Polyphasic Approach on Cyanobacterial Strains 125Nico Salmaso, Camilla Capelli, Rosmarie Rippka, and Annick Wilmotte SOP 4.2.1 Introduction 125 SOP 4.2.2 Experimental 126 SOP 4.2.3 References 131 5 Nucleic Acid Extraction 135Elke Dittmann, Anne Rantala-Ylinen, Vitor Ramos, Vitor Vasconcelos, Guntram Christiansen, and Rainer Kurmayer 5.1 Introduction 135 5.2 Specific Extraction Procedures and Storage 137 5.2.1 DNA Extraction from Laboratory Strains 137 5.2.2 DNA Extraction from Field Samples 137 5.2.3 DNA Extraction from Food Supplements 137 5.2.4 RNA Extraction from Laboratory Strains 138 5.2.5 RNA Extraction from Field Samples 138 5.2.6 Single Colony and Filament Analysis 138 5.2.7 Whole Genome Amplification 139 5.2.8 Nucleic Acid Storage 139 5.3 References 139 SOP 5.1 Standard DNA Isolation Technique for Cyanobacteria 140Elke Dittmann SOP 5.1.1 Introduction 140 SOP 5.1.2 Experimental 140 SOP 5.1.3 Procedure 141 SOP 5.1.4 Notes 141 SOP 5.1.5 References 142 SOP 5.2 DNA Isolation Protocol for Cyanobacteria with Extensive Mucilage 143Guntram Christiansen, Elisabeth Entfellner, and Rainer Kurmayer SOP 5.2.1 Introduction 143 SOP 5.2.2 Experimental 143 SOP 5.2.3 Procedure 144 SOP 5.2.4 Notes 145 SOP 5.2.5 References 145 SOP 5.3 Quantitative DNA Isolation from Filters 145Rainer Kurmayer SOP 5.3.1 Introduction 146 SOP 5.3.2 Experimental 146 SOP 5.3.3 Procedure 147 SOP 5.3.4 Notes 148 SOP 5.3.5 References 148 SOP 5.4 Genomic DNA Extraction from Single Filaments/Colonies for Multiple PCR Analyses 149Guntram Christiansen, Chen Qin, and Rainer Kurmayer SOP 5.4.1 Introduction 149 SOP 5.4.2 Experimental 149 SOP 5.4.3 Procedure 150 SOP 5.4.4 Notes 151 SOP 5.4.5 References 151 SOP 5.5 Whole Genome Amplification Using Bacteriophage Phi29 DNA Polymerase 151Guntram Christiansen and Rainer Kurmayer SOP 5.5.1 Introduction 151 SOP 5.5.2 Experimental 152 SOP 5.5.3 Procedure 152 SOP 5.5.4 Notes 152 SOP 5.5.5 Reference 153 SOP 5.6 DNA Extraction from Food Supplements 153Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos SOP 5.6.1 Introduction 153 SOP 5.6.2 Experimental 153 SOP 5.6.3 Procedure 154 SOP 5.6.4 Notes 155 SOP 5.6.5 References 156 SOP 5.7 RNA Extraction from Cyanobacteria 156Guntram Christiansen and Rainer Kurmayer SOP 5.7.1 Introduction 156 SOP 5.7.2 Experimental 156 SOP 5.7.3 Procedure 158 SOP 5.7.4 Notes 158 SOP 5.7.5 References 159 SOP 5.8 cDNA Synthesis 159Guntram Christiansen and Rainer Kurmayer SOP 5.8.1 Introduction 159 SOP 5.8.2 Experimental 159 SOP 5.8.3 Procedure 160 SOP 5.8.4 Notes 161 SOP 5.8.5 References 161 6 Conventional PCR 163Elke Dittmann, Anne Rantala-Ylinen, Kaarina Sivonen, Ilona Ga(2)ga(3)a, Joanna Mankiewicz-Boczek, Samuel Cires, Andreas Ballot, Guntram Christiansen, Rainer Kurmayer, Vitor Ramos, Vitor Vasconcelos, and Martin Saker 6.1 Introduction 163 6.2 Principle of PCR and Available Enzymes 164 6.2.1 Primer Development 165 6.2.2 Setup of PCR Conditions for DNA and Single Colony Analysis 168 6.2.3 Gel Electrophoresis and Documentation 168 6.2.4 Troubleshooting of PCR Results 168 6.2.5 PCR Product Downstream Processing (RFLP, Cloning, Sequencing) 169 6.3 Special Notes 170 6.4 References 170 SOP 6.1 PCR Detection of Microcystin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 172Elke Dittmann SOP 6.1.1 Introduction 172 SOP 6.1.2 Experimental 172 SOP 6.1.3 Procedure 173 SOP 6.1.4 Notes 174 SOP 6.1.5 Reference 174 SOP 6.2 PCR Detection of Microcystin and Nodularin Biosynthesis Genes in the Cyanobacterial Orders Oscillatoriales, Chroococcales, Stigonematales, and Nostocales 175Elke Dittmann, Joanna Mankiewicz-Boczek, and Ilona Ga(2)ga(3)a SOP 6.2.1 Introduction 175 SOP 6.2.2 Experimental 175 SOP 6.2.3 Procedure 177 SOP 6.2.4 Notes 177 SOP 6.2.5 References 178 SOP 6.3 Genus-Specific PCR Detection of Microcystin Biosynthesis Genes in Anabaena/Nodularia and Microcystis and Planktothrix, Respectively 179Anne Rantala-Ylinen and Kaarina Sivonen SOP 6.3.1 Introduction 179 SOP 6.3.2 Experimental 179 SOP 6.3.3 Procedure 181 SOP 6.3.4 Notes 181 SOP 6.3.5 References 181 SOP 6.4 PCR Detection of Anatoxin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 182Anne Rantala-Ylinen and Kaarina Sivonen SOP 6.4.1 Introduction 182 SOP 6.4.2 Experimental 182 SOP 6.4.3 Procedure 183 SOP 6.4.4 Notes 184 SOP 6.4.5 Reference 184 SOP 6.5 PCR Detection of the Saxitoxin Biosynthesis Genes, sxtA, sxtX, sxtH, sxtG, and sxtI 185Andreas Ballot and Samuel Cires SOP 6.5.1 Introduction 185 SOP 6.5.2 Experimental 187 SOP 6.5.3 Procedure 187 SOP 6.5.4 Notes 188 SOP 6.5.5 References 189 SOP 6.6 PCR Detection of the Cylindrospermopsin Biosynthesis Gene cyrJ 189Samuel Cires and Andreas Ballot SOP 6.6.1 Introduction 189 SOP 6.6.2 Experimental 190 SOP 6.6.3 Procedure 191 SOP 6.6.4 Notes 191 SOP 6.6.5 References 192 SOP 6.7 PCR from Single Filament of Toxigenic Planktothrix 193Qin Chen, Guntram Christiansen, and Rainer Kurmayer SOP 6.7.1 Introduction 193 SOP 6.7.2 Experimental 193 SOP 6.7.3 Procedure 194 SOP 6.7.4 Notes 195 SOP 6.7.5 References 195 SOP 6.8 Analysis of Microcystin Biosynthesis Gene Subpopulation Variability in Planktothrix 196Rainer Kurmayer SOP 6.8.1 Introduction 196 SOP 6.8.2 Experimental 196 SOP 6.8.3 Procedure 197 SOP 6.8.4 Notes 197 SOP 6.8.5 References 198 SOP 6.9 PCR Detection of Microcystin Biosynthesis Genes from Food Supplements 199Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos SOP 6.9.1 Introduction 199 SOP 6.9.2 Experimental 199 SOP 6.9.3 Procedure 201 SOP 6.9.4 Notes 202 SOP 6.9.5 References 203 7 Quantitative PCR 205Anne Rantala-Ylinen, Henna Savela, Kaarina Sivonen, and Rainer Kurmayer 7.1 Introduction 205 7.2 Primer/Probe Design 206 7.3 Optimization 208 7.4 Absolute Quantification 208 7.5 Relative Quantification 209 7.6 Calibration of qPCR Results 209 7.7 General Conclusions 210 7.8 References 210 SOP 7.1 Optimization of qPCR Assays 211Rainer Kurmayer SOP 7.1.1 Introduction 211 SOP 7.1.2 Experimental 211 SOP 7.1.3 Procedure 212 SOP 7.1.4 Notes 213 SOP 7.1.5 References 213 SOP 7.2 Calibration of qPCR Results 214Rainer Kurmayer SOP 7.2.1 Introduction 214 SOP 7.2.2 Experimental 214 SOP 7.2.3 Procedure 215 SOP 7.2.4 Notes 217 SOP 7.2.5 References 217 SOP 7.3 Quantification of Potentially Microcystin/Nodularin-Producing Anabaena, Microcystis, Planktothrix, and Nodularia 218Anne Rantala-Ylinen, Kaarina Sivonen, and Rainer Kurmayer SOP 7.3.1 Introduction 218 SOP 7.3.2 Experimental 219 SOP 7.3.3 Procedure 219 SOP 7.3.4 Notes 221 SOP 7.3.5 References 221 SOP 7.4 Relative Quantification of Microcystis or Planktothrix mcy Genotypes Using qPCR 222Rainer Kurmayer SOP 7.4.1 Introduction 222 SOP 7.4.2 Experimental 222 SOP 7.4.3 Procedure 224 SOP 7.4.4 Notes 225 SOP 7.4.5 References 225 SOP 7.5 Quantification of Transcript Amounts of mcy Genes in Planktothrix 226Guntram Christiansen and Rainer Kurmayer SOP 7.5.1 Introduction 226 SOP 7.5.2 Experimental 226 SOP 7.5.3 Procedure 227 SOP 7.5.4 Notes 228 SOP 7.5.5 References 228 SOP 7.6 Quantification of Potentially Cylindrospermopsin-Producing Chrysosporum ovalisporum 229Rehab El-Shehawy and Antonio Quesada SOP 7.6.1 Introduction 229 SOP 7.6.2 Experimental 229 SOP 7.6.3 Procedure 230 SOP 7.6.4 Notes 231 SOP 7.6.5 References 231 SOP 7.7 qPCR Detection of the Paralytic Shellfish Toxin Biosynthesis Gene sxtB 231Henna Savela SOP 7.7.1 Introduction 231 SOP 7.7.2 Experimental 232 SOP 7.7.3 Procedure 233 SOP 7.7.4 Notes 234 SOP 7.7.5 References 234 SOP 7.8 Application of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines to Quantitative Analysis of Toxic Cyanobacteria 234Henna Savela SOP 7.8.1 Introduction 234 SOP 7.8.2 Sampling 235 SOP 7.8.3 Sample Preparation and DNA Extraction 235 SOP 7.8.4 Target Information and Oligonucleotide Design 235 SOP 7.8.5 qPCR Protocol 238 SOP 7.8.6 qPCR Validation 239 SOP 7.8.7 Data Analysis 239 SOP 7.8.8 Reference 239 8 DNA (Diagnostic) and cDNA Microarray 241Anne Rantala-Ylinen, Kaarina Sivonen, and Annick Wilmotte 8.1 DNA (Diagnostic) Microarray 241 8.1.1 Introduction 241 8.1.2 Methodological Principles 242 8.1.3 General Conclusions 243 8.1.4 References 243 8.2 cDNA Microarray for Cyanobacteria 244Hans C.P. Matthijs and J. Merijn Schuurmans 8.2.1 Introduction 244 8.2.2 Principles of Microarray Use 244 8.2.3 Considerations for Experimental Design 245 8.2.4 Microarray: Practical Approach 246 8.2.5 Microarray: Data Analysis 246 8.2.6 References 246 SOP 8.1 DNA-Chip Detection of Potential Microcystin and Nodularin Producing Cyanobacteria in Environmental Water Samples 248Anne Rantala-Ylinen and Kaarina Sivonen SOP 8.1.1 Introduction 248 SOP 8.1.2 Experimental 249 SOP 8.1.3 Procedure 250 SOP 8.1.4 Notes 253 SOP 8.1.5 References 253 SOP 8.2 cDNA Microarrays for Cyanobacteria 254J. Merijn Schuurmans and Hans C.P. Matthijs SOP 8.2.1 Introduction 254 SOP 8.2.2 Experimental 254 SOP 8.2.3 Procedure 256 SOP 8.2.4 Notes 259 SOP 8.2.5 Reference 261 9 Analysis of Toxigenic Cyanobacterial Communities through Denaturing Gradient Gel Electrophoresis 263Iwona Jasser, Aleksandra Bukowska, Jean-Francois Humbert, Kaisa Haukka, and David P. Fewer 9.1 Introduction 263 9.2 Main Applications of the Method 264 9.3 Possible Applications 264 9.4 DGGE Procedure 265 9.5 General Conclusions Including Pros and Cons of the Method 267 9.6 Optimization of the Method and Troubleshooting 267 9.7 References 268 SOP 9.1 DGGE-mcyA Conditions 270Aleksandra Bukowska and Iwona Jasser SOP 9.1.1 Introduction 270 SOP 9.1.2 Experimental 270 SOP 9.1.3 Procedure 272 SOP 9.1.4 Notes 275 SOP 9.1.5 References 275 10 Monitoring of Toxigenic Cyanobacteria Using Next-Generation Sequencing Techniques 277Li Deng, Maxime Sweetlove, Stephan Blank, Dagmar Obbels, Elie Verleyen, Wim Vyverman, and Rainer Kurmayer 10.1 Introduction 277 10.2 Specific Procedures 279 10.2.1 16S rRNA Gene Amplicon Library Preparation 279 10.2.2 Amplicon Purification, Quantification and Pooling 280 10.2.3 Sequencing 280 10.2.4 Bioinformatic Exploration of Sequencing Results 281 10.2.5 General Conclusions Including Pros and Cons of the Method 281 10.2.6 References 281 10.3 Bioinformatic Processing of Amplicon Sequencing Datasets 283Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim Vyverman 10.3.1 Introduction 283 10.3.2 Sequencing Platforms 283 10.3.3 Data Formats 284 10.3.4 Error Associated with NGS Data 285 10.3.5 OTU Delineation: Choosing a Similarity Threshold 286 10.3.6 Conclusions 286 10.4 References 286 SOP 10.1 Standard Technique to Generating 16S rRNA PCR Amplicons for NGS 288Li Deng, Stephan Blank, Guntram Christiansen, and Rainer Kurmayer SOP 10.1.1 Introduction 288 SOP 10.1.2 Experimental 288 SOP 10.1.3 Procedure 289 SOP 10.1.4 Notes 290 SOP 10.1.5 References 290 SOP 10.2 Bioinformatics Analysis for NGS Amplicon Sequencing 291Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim Vyverman SOP 10.2.1 Introduction 291 SOP 10.2.2 Experimental 291 SOP 10.2.3 Practical Tips and Alternatives for Quality Filtering 298 SOP 10.2.4 References 298 11 Application of Molecular Tools in Monitoring Cyanobacteria and Their Potential Toxin Production 301Vitor Ramos, Cristiana Moreira, Joanna Mankiewicz-Boczek, and Vitor Vasconcelos 11.1 Introduction 301 11.2 Possible Applications 303 11.3 Checklist of Publications, Applications and Lessons from Practice 315 11.3.1 Molecular-Based Studies on (Toxic) Cyanobacteria: Overview of Methods Being Used, and Generic Findings and Concerns 315 11.3.2 The Need for Complementary Approaches 316 11.3.3 Interpreting Results 316 11.3.4 Choice of Molecular Tools for Toxigenicity Assessment 317 11.3.5 Common and Possible Applications of Molecular Tools 318 11.4 General Conclusions 321 11.5 Acknowledgments 324 11.6 References 324 Appendix: Supplementary Tables 335 Cyanobacterial Species Cited in the Book 376 Glossary 379 Index 393