As a result of key issues that certain Vibrio specie, especially Vibrio harveyi, may cause the aquaculture industries a rapid method to identify Vibrio isolates is needed. Early diagnosis of a V. harveyi infection can aid disease surveillance, cure and prevention in cultured marine animals. As a result, the use of PCR to assist in the identification of Vibrio is increasing and a method of extracting DNA in an inexpensive, quick and simple way is also of an important requirement to facilitate rapid diagnosis. This document comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus. Strains were analyzed for adherence to a Hep-2 cell line. Four different DNA extraction approaches were examined and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined…
Contents: Detection and characterisation of Vibrio harveyi isolates
1.0 INTRODUCTION
1.1: Vibrionaceae
1.2. Reservoir and transmission
1.3: Vibrio species as fish pathogens
1.4: Clinical features of Vibrio infection in aquatic animals
1.5: Virulence factors of Vibrio species
1.6: Treatment
1.7: Isolation and identification of Vibrio species
2.0 MATERIAL AND METHODS
2.1 Material
2.1.1: Bacterial isolates
2.1.2: Growth media
2.1.3: Apparatus
2.1.4: Equipment
2.1.5: Biochemical identification of bacterial isolates
2.1.6: DNA extraction
2.1.6.i: Phenol: chloroform extraction
2.1.6.ii: High-Pure PCR Template Preparation Kit
2.1.7: Agarose Gel Electrophoresis
2.1.8: PCR (polymerase chain reaction)
2.2 Methods
2.2.1: Biochemical characterisation
2.2.1.i: General biochemical characterisation
2.2.1.ii: Comparison of Vibrio growth on four different media
2.2.1.iii: API 20E, biochemical characterisation
2.2.2: Tissue adherence test
2.2.3: DNA extraction
2.2.3.i: Phenol: chloroform extraction according to Montes et al., 2003
2.2.3.ii: High-Pure PCR Template Preparation Kit
2.2.3.iii: Boiling
2.2.3.iv: Microwaving
2.2.4: DNA quantification
2.2.5: Agarose Gel Electrophoresis
2.2.5.i: Visualisation of extracted DNA
2.2.5.ii: Visualisation of PCR-products
2.2.6: PCR (polymerase chain reaction)
2.2.6.i: [MgCl2] optimization
2.2.6.ii: 16S rDNA sequence determination
2.2.6.iii: VH Amplification
2.2.6.iv: Diagnostic Detection Limit
2.2.6.v: Analytical Sensitivity
3.0 RESULTS
3.1: Phenotypic identification
3.1.1: Morphology
3.1.3: Tissue adherence test
3.2: Biochemical characteristics
3.2.1: General characteristics
3.2.2: API E20, biochemical characterisation
3.3: DNA extraction
3.3.1: Visualisation of genomic DNA from the four different extracting
methods using gel electrophoresis
3.4: 16S rDNA gene determination
3.4.1: [MgCl2] optimization
3.4.2: Amplicon determination using the 16S rDNA-primers
3.5: PCR using V. harveyi VH-1 and VH-2 primers
3.5.1: [MgCl2] optimization
3.5.2: Amplicon determination using VH-primers
3.6 Diagnostic Detection Limit
3.7 Analytical Sensitivity…
Source: Uppsala University Library
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