PhD Thesis MJProl .pdf - digital-csic Digital CSIC - Consejo Superior ...

xii Index
4.2.4. Mixing of samples and DNA extraction 59
4.2.5. Design of primers for quantitative real-time PCR 59
4.2.6. Quantitative real-time PCR
4.2.7. Standard curves and quantification of strain mixtures using
61
quantitative real-time PCR
4.2.8. Quantification by real-time PCR of bacteria experimentally
introduced in rotifers and transferred to seawater and turbot
62
larvae 62
4.3. RESULTS 63
4.3.1. Verification of primers specificity 63
4.3.2. Standard curves for quantitative real-time PCR
4.3.3. Real-time PCR quantification of known concentrations of the
64
target bacteria
4.3.4. Real-time PCR quantification of target bacteria experimentally
introduced in rotifers (bioencapsulation) and transferred to
66
seawater and to turbot larvae in a challenge trial 68
4.4. DISCUSSION 69
4.5. REFERENCES 72
5. CHAPTER II:
DIFFERENT COLONIZATION AND RESIDENCE TIME OF
LISTONELLA ANGUILLARUM AND VIBRIO SPLENDIDUS IN THE
ROTIFER BRACHIONUS PLICATILIS DETERMINED BY REAL-
TIME PCR AND DGGE 79
5.1. INTRODUCTION 79
5.2. MATERIALS AND METHODS 81
5.2.1. Bacterial strains and culture conditions 81
5.2.2. Infection of rotifers with the pathogens 81
5.2.3. Preparation of samples for microbiological analysis 82
5.2.4. DNA extraction 83
5.2.5. Quantitative real-time PCR 83
5.2.6. PCR-DGGE 84
5.2.7. Analysis of DGGE profiles 85
5.2.8. Sequencing of DNA from PCR fragments and bacterial isolates 86
5.3. RESULTS
5.3.1. Colonization and residence time of Listonella anguillarum 90-
87
11-287 and Vibrio splendidus DMC-1 in rotifers 87