Molecular Tools for the Detection and Quantification of Toxigenic Cyanobacteria

EDITED BYRAINER KURMAYER, PhD, is an Associate Professor at the University of Innsbruck, Research Institute for Limnology, Mondsee, Austria. KAARINA SIVONEN, PhD, is a Professor of Microbiology at the University of Helsinki, Finland. ANNICK WILMOTTE, PhD, is a FRS-FNRS Research Associate at InBios – Center for Protein Engineering, University of Liège, Belgium. NICO SALMASO, PhD, is Head of the Hydrobiology Unit of the Istituto Agrario di S. Michele All'Adige, Fondazione E. Mach (FEM), Trento, Italy.

• List of Contributors xixAbout the Editors xxiiiAbout the Book xxviiPreface xxixAcknowledgments xxxi1 Introduction 1Rainer Kurmayer, Kaarina Sivonen, and Nico Salmaso1.1 A Brief Historical Overview 11.2 The Genetic Basis of Toxin Production 21.2.1 Microcystin and Nodularin 21.2.2 Cylindrospermopsin 51.2.3 Saxitoxin 61.2.4 Anatoxin 81.3 Application of Molecular Tools 81.4 Laboratory Safety Issues 131.5 References 142 Sampling and Metadata 19Rainer Kurmayer, Guntram Christiansen, Konstantinos Kormas, Wim Vyverman, Elie Verleyen, Vitor Ramos, Vitor Vasconcelos, and Nico Salmaso2.1 Introduction 192.2 Handling of Samples 202.3 Sample Contamination 212.4 Sampling 212.4.1 Quantitative Depth-Integrated and Discrete Sampling 212.4.2 Qualitative Plankton Net Sampling 222.4.3 Surface (Scum Material) Sampling 222.4.4 Benthic (Terrestrial) Cyanobacteria Sampling 222.4.5 Food Supplement Sampling 222.4.6 Isolation of Single Colonies/Filaments 222.5 Subsampling Food Supplement Samples 232.6 Sampling of Nucleic Acids 232.7 General Conclusions 242.8 References 24SOP 2.1 Sampling and Filtration (DNA) 26Rainer Kurmayer and Konstantinos KormasSOP 2.1.1 Introduction 26SOP 2.1.2 Experimental 26SOP 2.1.3 Procedure 27SOP 2.1.4 Notes 28SOP 2.1.5 References 29SOP 2.2 Sampling of Benthic Cyanobacteria 29Wim Vyverman and Elie VerleyenSOP 2.2.1 Introduction 29SOP 2.2.2 Experimental 30SOP 2.2.3 Procedure 30SOP 2.2.4 Notes 31SOP 2.2.5 References 31SOP 2.3 Isolation of Single Cyanobacteria Colonies/Filaments 32Rainer KurmayerSOP 2.3.1 Introduction 32SOP 2.3.2 Experimental 32SOP 2.3.3 Procedure 33SOP 2.3.4 Notes 33SOP 2.3.5 References 33SOP 2.4 Sampling Food Supplements 34Vitor Ramos, Cristiana Moreira, and Vitor VasconcelosSOP 2.4.1 Introduction 34SOP 2.4.2 Experimental 35SOP 2.4.3 Procedure (Fig. 8.3) 35SOP 2.4.4 Notes 36SOP 2.4.5 References 36SOP 2.5 Sampling and Filtration (RNA) 37Rainer Kurmayer and Guntram ChristiansenSOP 2.5.1 Introduction 37SOP 2.5.2 Experimental 37SOP 2.5.3 Procedure 38SOP 2.5.4 Notes 38SOP 2.5.5 References 38SOP 2.6 Sampling of Abiotic and Biotic Data and Recording Metadata 39Elie Verleyen, Maxime Sweetlove, Dagmar Obbels, and Wim VyvermanSOP 2.6.1 Introduction 39SOP 2.6.2 Experimental 39SOP 2.6.3 Type of Metadata and Additional Biotic and Abiotic Data 40SOP 2.6.4 Notes 41SOP 2.6.5 References 423 Isolation, Purification, and Cultivation of Toxigenic Cyanobacteria 43Sigrid Haande, Iwona Jasser, Muriel Gugger, Camilla H.C. Hagman, Annick Wilmotte, and Andreas Ballot3.1 Introduction 433.2 Methodical Principles for Cyanobacterial Isolation, Purification, and Cultivation 443.2.1 Sampling, Identification, and Treatments Prior to the Isolation of Cyanobacteria 443.2.2 Traditional Techniques for the Isolation and Purification of Cyanobacteria 453.2.3 Culture Media Preparation 473.2.4 Cultivation Conditions 483.3 General Conclusions 493.4 References 49SOP 3.1 Isolation, Purification, and Clonal Isolate Testing 51Sigrid Haande, Camilla H.C. Hagman, and Andreas BallotSOP 3.1.1 Introduction 51SOP 3.1.2 Experimental 51SOP 3.1.3 Procedure 52SOP 3.1.4 Notes 54SOP 3.1.5 References 54SOP 3.2 Isolation of Picocyanobacterial Cells by Flow Cytometer (FCM) Sorting 55Ewa Koz³owska and Iwona JasserSOP 3.2.1 Introduction 55SOP 3.2.2 Experimental 56SOP 3.2.3 Procedure 56SOP 3.2.4 Notes 58SOP 3.2.5 References 59SOP 3.3 Axenization 60Muriel GuggerSOP 3.3.1 Introduction 60SOP 3.3.2 Experimental 60SOP 3.3.3 Procedure 61SOP 3.3.4 Notes 63SOP 3.3.5 References 63SOP 3.4 Culture Media (Solid and Liquid) 64Sigrid Haande, Camilla H.C. Hagman, and Andreas BallotSOP 3.4.1 Introduction 64SOP 3.4.2 Experimental 64SOP 3.4.3 Procedure 65SOP 3.4.4 Notes 68SOP 3.4.5 References 68SOP 3.5 Strain Maintenance (Living Cultures) 69Sigrid Haande, Camilla H.C. Hagman, and Andreas BallotSOP 3.5.1 Introduction 69SOP 3.5.2 Experimental 69SOP 3.5.3 Procedure 70SOP 3.5.4 Notes 72SOP 3.5.5 References 73SOP 3.6 Cryopreservation and Recovery 73Muriel GuggerSOP 3.6.1 Introduction 73SOP 3.6.2 Experimental 74SOP 3.6.3 Procedure 75SOP 3.6.4 Notes 78SOP 3.6.5 References 784 Taxonomic Identification of Cyanobacteria by a Polyphasic Approach 79Annick Wilmotte, H. Dail Laughinghouse IV, Camilla Capelli, Rosmarie Rippka, and Nico Salmaso4.1 Introduction 794.2 Nomenclature and Classification of Cyanobacteria 824.3 Microscopy 844.3.1 Light Microscopy 844.3.2 Autofluorescence Microscopy 864.4 Molecular Markers: Single Loci 874.5 Molecular Markers: Multiple Loci 944.5.1 Multilocus Sequence Typing (MLST) and Multilocus Sequence Analysis (MLSA) 944.5.2 Genome-Based Extension of MLST and MLSA 964.6 Molecular Typing Methods Based on Gel Electrophoresis 964.7 Denaturing Gradient Gel Electrophoresis (DGGE) 974.8 Taxonomic and Molecular Databases 974.9 The Polyphasic Approach 984.10 Final Considerations 1054.11 References 106SOP 4.1 Taxonomic Identification by Light Microscopy 120Nico Salmaso, Rosmarie Rippka, and Annick WilmotteSOP 4.1.1 Introduction 120SOP 4.1.2 Experimental 121SOP 4.1.3 References 124SOP 4.2 Polyphasic Approach on Cyanobacterial Strains 125Nico Salmaso, Camilla Capelli, Rosmarie Rippka, and Annick WilmotteSOP 4.2.1 Introduction 125SOP 4.2.2 Experimental 126SOP 4.2.3 References 1315 Nucleic Acid Extraction 135Elke Dittmann, Anne Rantala-Ylinen, Vitor Ramos, Vitor Vasconcelos, Guntram Christiansen, and Rainer Kurmayer5.1 Introduction 1355.2 Specific Extraction Procedures and Storage 1375.2.1 DNA Extraction from Laboratory Strains 1375.2.2 DNA Extraction from Field Samples 1375.2.3 DNA Extraction from Food Supplements 1375.2.4 RNA Extraction from Laboratory Strains 1385.2.5 RNA Extraction from Field Samples 1385.2.6 Single Colony and Filament Analysis 1385.2.7 Whole Genome Amplification 1395.2.8 Nucleic Acid Storage 1395.3 References 139SOP 5.1 Standard DNA Isolation Technique for Cyanobacteria 140Elke DittmannSOP 5.1.1 Introduction 140SOP 5.1.2 Experimental 140SOP 5.1.3 Procedure 141SOP 5.1.4 Notes 141SOP 5.1.5 References 142SOP 5.2 DNA Isolation Protocol for Cyanobacteria with Extensive Mucilage 143Guntram Christiansen, Elisabeth Entfellner, and Rainer KurmayerSOP 5.2.1 Introduction 143SOP 5.2.2 Experimental 143SOP 5.2.3 Procedure 144SOP 5.2.4 Notes 145SOP 5.2.5 References 145SOP 5.3 Quantitative DNA Isolation from Filters 145Rainer KurmayerSOP 5.3.1 Introduction 146SOP 5.3.2 Experimental 146SOP 5.3.3 Procedure 147SOP 5.3.4 Notes 148SOP 5.3.5 References 148SOP 5.4 Genomic DNA Extraction from Single Filaments/Colonies for Multiple PCR Analyses 149Guntram Christiansen, Chen Qin, and Rainer KurmayerSOP 5.4.1 Introduction 149SOP 5.4.2 Experimental 149SOP 5.4.3 Procedure 150SOP 5.4.4 Notes 151SOP 5.4.5 References 151SOP 5.5 Whole Genome Amplification Using Bacteriophage Phi29 DNA Polymerase 151Guntram Christiansen and Rainer KurmayerSOP 5.5.1 Introduction 151SOP 5.5.2 Experimental 152SOP 5.5.3 Procedure 152SOP 5.5.4 Notes 152SOP 5.5.5 Reference 153SOP 5.6 DNA Extraction from Food Supplements 153Vitor Ramos, Cristiana Moreira, and Vitor VasconcelosSOP 5.6.1 Introduction 153SOP 5.6.2 Experimental 153SOP 5.6.3 Procedure 154SOP 5.6.4 Notes 155SOP 5.6.5 References 156SOP 5.7 RNA Extraction from Cyanobacteria 156Guntram Christiansen and Rainer KurmayerSOP 5.7.1 Introduction 156SOP 5.7.2 Experimental 156SOP 5.7.3 Procedure 158SOP 5.7.4 Notes 158SOP 5.7.5 References 159SOP 5.8 cDNA Synthesis 159Guntram Christiansen and Rainer KurmayerSOP 5.8.1 Introduction 159SOP 5.8.2 Experimental 159SOP 5.8.3 Procedure 160SOP 5.8.4 Notes 161SOP 5.8.5 References 1616 Conventional PCR 163Elke Dittmann, Anne Rantala-Ylinen, Kaarina Sivonen, Ilona Ga²ga³a, Joanna Mankiewicz-Boczek, Samuel Cirés, Andreas Ballot, Guntram Christiansen, Rainer Kurmayer, Vitor Ramos, Vitor Vasconcelos, and Martin Saker6.1 Introduction 1636.2 Principle of PCR and Available Enzymes 1646.2.1 Primer Development 1656.2.2 Setup of PCR Conditions for DNA and Single Colony Analysis 1686.2.3 Gel Electrophoresis and Documentation 1686.2.4 Troubleshooting of PCR Results 1686.2.5 PCR Product Downstream Processing (RFLP, Cloning, Sequencing) 1696.3 Special Notes 1706.4 References 170SOP 6.1 PCR Detection of Microcystin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 172Elke DittmannSOP 6.1.1 Introduction 172SOP 6.1.2 Experimental 172SOP 6.1.3 Procedure 173SOP 6.1.4 Notes 174SOP 6.1.5 Reference 174SOP 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 Ga²ga³aSOP 6.2.1 Introduction 175SOP 6.2.2 Experimental 175SOP 6.2.3 Procedure 177SOP 6.2.4 Notes 177SOP 6.2.5 References 178SOP 6.3 Genus-Specific PCR Detection of Microcystin Biosynthesis Genes in Anabaena/Nodularia and Microcystis and Planktothrix, Respectively 179Anne Rantala-Ylinen and Kaarina SivonenSOP 6.3.1 Introduction 179SOP 6.3.2 Experimental 179SOP 6.3.3 Procedure 181SOP 6.3.4 Notes 181SOP 6.3.5 References 181SOP 6.4 PCR Detection of Anatoxin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 182Anne Rantala-Ylinen and Kaarina SivonenSOP 6.4.1 Introduction 182SOP 6.4.2 Experimental 182SOP 6.4.3 Procedure 183SOP 6.4.4 Notes 184SOP 6.4.5 Reference 184SOP 6.5 PCR Detection of the Saxitoxin Biosynthesis Genes, sxtA, sxtX, sxtH, sxtG, and sxtI 185Andreas Ballot and Samuel CirésSOP 6.5.1 Introduction 185SOP 6.5.2 Experimental 187SOP 6.5.3 Procedure 187SOP 6.5.4 Notes 188SOP 6.5.5 References 189SOP 6.6 PCR Detection of the Cylindrospermopsin Biosynthesis Gene cyrJ 189Samuel Cirés and Andreas BallotSOP 6.6.1 Introduction 189SOP 6.6.2 Experimental 190SOP 6.6.3 Procedure 191SOP 6.6.4 Notes 191SOP 6.6.5 References 192SOP 6.7 PCR from Single Filament of Toxigenic Planktothrix 193Qin Chen, Guntram Christiansen, and Rainer KurmayerSOP 6.7.1 Introduction 193SOP 6.7.2 Experimental 193SOP 6.7.3 Procedure 194SOP 6.7.4 Notes 195SOP 6.7.5 References 195SOP 6.8 Analysis of Microcystin Biosynthesis Gene Subpopulation Variability in Planktothrix 196Rainer KurmayerSOP 6.8.1 Introduction 196SOP 6.8.2 Experimental 196SOP 6.8.3 Procedure 197SOP 6.8.4 Notes 197SOP 6.8.5 References 198SOP 6.9 PCR Detection of Microcystin Biosynthesis Genes from FoodSupplements 199Vitor Ramos, Cristiana Moreira, and Vitor VasconcelosSOP 6.9.1 Introduction 199SOP 6.9.2 Experimental 199SOP 6.9.3 Procedure 201SOP 6.9.4 Notes 202SOP 6.9.5 References 2037 Quantitative PCR 205Anne Rantala-Ylinen, Henna Savela, Kaarina Sivonen, and Rainer Kurmayer7.1 Introduction 2057.2 Primer/Probe Design 2067.3 Optimization 2087.4 Absolute Quantification 2087.5 Relative Quantification 2097.6 Calibration of qPCR Results 2097.7 General Conclusions 2107.8 References 210SOP 7.1 Optimization of qPCR Assays 211Rainer KurmayerSOP 7.1.1 Introduction 211SOP 7.1.2 Experimental 211SOP 7.1.3 Procedure 212SOP 7.1.4 Notes 213SOP 7.1.5 References 213SOP 7.2 Calibration of qPCR Results 214Rainer KurmayerSOP 7.2.1 Introduction 214SOP 7.2.2 Experimental 214SOP 7.2.3 Procedure 215SOP 7.2.4 Notes 217SOP 7.2.5 References 217SOP 7.3 Quantification of Potentially Microcystin/Nodularin-Producing Anabaena, Microcystis, Planktothrix, and Nodularia 218Anne Rantala-Ylinen, Kaarina Sivonen, and Rainer KurmayerSOP 7.3.1 Introduction 218SOP 7.3.2 Experimental 219SOP 7.3.3 Procedure 219SOP 7.3.4 Notes 221SOP 7.3.5 References 221SOP 7.4 Relative Quantification of Microcystis or Planktothrix mcy Genotypes Using qPCR 222Rainer KurmayerSOP 7.4.1 Introduction 222SOP 7.4.2 Experimental 222SOP 7.4.3 Procedure 224SOP 7.4.4 Notes 225SOP 7.4.5 References 225SOP 7.5 Quantification of Transcript Amounts of mcy Genes in Planktothrix 226Guntram Christiansen and Rainer KurmayerSOP 7.5.1 Introduction 226SOP 7.5.2 Experimental 226SOP 7.5.3 Procedure 227SOP 7.5.4 Notes 228SOP 7.5.5 References 228SOP 7.6 Quantification of Potentially Cylindrospermopsin-Producing Chrysosporum ovalisporum 229Rehab El-Shehawy and Antonio QuesadaSOP 7.6.1 Introduction 229SOP 7.6.2 Experimental 229SOP 7.6.3 Procedure 230SOP 7.6.4 Notes 231SOP 7.6.5 References 231SOP 7.7 qPCR Detection of the Paralytic Shellfish Toxin Biosynthesis Gene sxtB 231Henna SavelaSOP 7.7.1 Introduction 231SOP 7.7.2 Experimental 232SOP 7.7.3 Procedure 233SOP 7.7.4 Notes 234SOP 7.7.5 References 234SOP 7.8 Application of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines to Quantitative Analysis of Toxic Cyanobacteria 234Henna SavelaSOP 7.8.1 Introduction 234SOP 7.8.2 Sampling 235SOP 7.8.3 Sample Preparation and DNA Extraction 235SOP 7.8.4 Target Information and Oligonucleotide Design 235SOP 7.8.5 qPCR Protocol 238SOP 7.8.6 qPCR Validation 239SOP 7.8.7 Data Analysis 239SOP 7.8.8 Reference 2398 DNA (Diagnostic) and cDNA Microarray 241Anne Rantala-Ylinen, Kaarina Sivonen, and Annick Wilmotte8.1 DNA (Diagnostic) Microarray 2418.1.1 Introduction 2418.1.2 Methodological Principles 2428.1.3 General Conclusions 2438.1.4 References 2438.2 cDNA Microarray for Cyanobacteria 244Hans C.P. Matthijs and J. Merijn Schuurmans8.2.1 Introduction 2448.2.2 Principles of Microarray Use 2448.2.3 Considerations for Experimental Design 2458.2.4 Microarray: Practical Approach 2468.2.5 Microarray: Data Analysis 2468.2.6 References 246SOP 8.1 DNA-Chip Detection of Potential Microcystin and Nodularin Producing Cyanobacteria in Environmental Water Samples 248Anne Rantala-Ylinen and Kaarina SivonenSOP 8.1.1 Introduction 248SOP 8.1.2 Experimental 249SOP 8.1.3 Procedure 250SOP 8.1.4 Notes 253SOP 8.1.5 References 253SOP 8.2 cDNA Microarrays for Cyanobacteria 254J. Merijn Schuurmans and Hans C.P. MatthijsSOP 8.2.1 Introduction 254SOP 8.2.2 Experimental 254SOP 8.2.3 Procedure 256SOP 8.2.4 Notes 259SOP 8.2.5 Reference 2619 Analysis of Toxigenic Cyanobacterial Communities through Denaturing Gradient Gel Electrophoresis 263Iwona Jasser, Aleksandra Bukowska, Jean-Francois Humbert, Kaisa Haukka, and David P. Fewer9.1 Introduction 2639.2 Main Applications of the Method 2649.3 Possible Applications 2649.4 DGGE Procedure 2659.5 General Conclusions Including Pros and Cons of the Method 2679.6 Optimization of the Method and Troubleshooting 2679.7 References 268SOP 9.1 DGGE-mcyA Conditions 270Aleksandra Bukowska and Iwona JasserSOP 9.1.1 Introduction 270SOP 9.1.2 Experimental 270SOP 9.1.3 Procedure 272SOP 9.1.4 Notes 275SOP 9.1.5 References 27510 Monitoring of Toxigenic Cyanobacteria Using Next-Generation Sequencing Techniques 277Li Deng, Maxime Sweetlove, Stephan Blank, Dagmar Obbels, Elie Verleyen, Wim Vyverman, and Rainer Kurmayer10.1 Introduction 27710.2 Specific Procedures 27910.2.1 16S rRNA Gene Amplicon Library Preparation 27910.2.2 Amplicon Purification, Quantification and Pooling 28010.2.3 Sequencing 28010.2.4 Bioinformatic Exploration of Sequencing Results 28110.2.5 General Conclusions Including Pros and Cons of the Method 28110.2.6 References 28110.3 Bioinformatic Processing of Amplicon Sequencing Datasets 283Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim Vyverman10.3.1 Introduction 28310.3.2 Sequencing Platforms 28310.3.3 Data Formats 28410.3.4 Error Associated with NGS Data 28510.3.5 OTU Delineation: Choosing a Similarity Threshold 28610.3.6 Conclusions 28610.4 References 286SOP 10.1 Standard Technique to Generating 16S rRNA PCR Amplicons for NGS 288Li Deng, Stephan Blank, Guntram Christiansen, and Rainer KurmayerSOP 10.1.1 Introduction 288SOP 10.1.2 Experimental 288SOP 10.1.3 Procedure 289SOP 10.1.4 Notes 290SOP 10.1.5 References 290SOP 10.2 Bioinformatics Analysis for NGS Amplicon Sequencing 291Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim VyvermanSOP 10.2.1 Introduction 291SOP 10.2.2 Experimental 291SOP 10.2.3 Practical Tips and Alternatives for Quality Filtering 298SOP 10.2.4 References 29811 Application of Molecular Tools in Monitoring Cyanobacteria and Their Potential Toxin Production 301Vitor Ramos, Cristiana Moreira, Joanna Mankiewicz-Boczek, and Vitor Vasconcelos11.1 Introduction 30111.2 Possible Applications 30311.3 Checklist of Publications, Applications and Lessons from Practice 31511.3.1 Molecular-Based Studies on (Toxic) Cyanobacteria: Overview of Methods Being Used, and Generic Findings and Concerns 31511.3.2 The Need for Complementary Approaches 31611.3.3 Interpreting Results 31611.3.4 Choice of Molecular Tools for Toxigenicity Assessment 31711.3.5 Common and Possible Applications of Molecular Tools 31811.4 General Conclusions 32111.5 Acknowledgments 32411.6 References 324Appendix: Supplementary Tables 335Cyanobacterial Species Cited in the Book 376Glossary 379Index 393