Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis
June 17th, 2019
The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay presented here enables inexpensive, robust and portable high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. Starting from pure cultures, the assay employs multiplex PCR and capillary electrophoresis to produce ten-loci MLVA profiles for downstream applications.
Overview
The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay enables high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. This method is cost-effective, robust, and portable, allowing for improved diagnostic specificity and infection tracing.
Key Study Components
Area of Science
- Microbiology
- Genotyping
- Pathogen Epidemiology
Background
- Yersinia ruckeri is a significant fish pathogen.
- Understanding its epidemiology is crucial for aquaculture.
- Existing typing schemes lacked high strain resolution.
- The MLVA assay offers a solution to these limitations.
Purpose of Study
- To develop a high-resolution genotyping method for Yersinia ruckeri.
- To enhance diagnostic capabilities for infection tracing.
- To facilitate comparisons across laboratories.
Methods Used
- Multiplex PCR for DNA amplification.
- Capillary electrophoresis for fragment analysis.
- Use of ultra-purified water for DNA resuspension.
- Application of Gene Mapper software for size calling.
Main Results
- MLVA provides high strain resolution for Yersinia ruckeri.
- Improved diagnostic specificity allows for better infection tracing.
- Portable results facilitate inter-laboratory comparisons.
- Distinction between virulent and non-virulent strains is achievable.
Conclusions
- The MLVA assay is a valuable tool for studying Yersinia ruckeri.
- It enhances our understanding of pathogen epidemiology.
- This method can be easily implemented in various laboratories.
Frequently Asked Questions
Executive Industry Relevance
High-resolution genotyping of fish pathogens like Yersinia ruckeri supports epizootiological investigations and infection tracing in aquaculture. Portable, comparable MLVA profiles enable rapid strain discrimination and cross-laboratory data sharing. This enhances predictive confidence in pathogen surveillance and informs risk-adjusted decisions for vaccine development and outbreak management.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables discrimination of virulent from non-virulent Yersinia ruckeri clones, supporting target validation of virulence-associated factors.
- Operational Value: Provides unambiguous, numerical MLVA profiles for reproducible strain identification across laboratories.
Screening & Assay Development
- Scientific Value: Ten-locus VNTR analysis delivers high strain resolution for precise isolate discrimination in screening campaigns.
- Operational Value: Multiplex PCR and capillary electrophoresis allow standardized, high-throughput processing of up to 48 isolates per day.
Translational & Preclinical Research
- Scientific Value: MLVA-derived cluster analysis links strain genotypes to geographic or farm origins, supporting translational epidemiology.
- Operational Value: Portable numerical outputs facilitate integration into bioinformatics pipelines for Minimum spanning tree generation and comparative analysis.
Pipeline & Workflow Integration
The MLVA workflow fits within discovery biology to preclinical transition, enabling hypothesis testing of pathogen spread and informing lead identification for intervention strategies.
- Discovery Biology: Supports interrogation of therapeutic hypotheses by resolving population structure and clonal dynamics of Yersinia ruckeri.
- Screening: Delivers quantitative, reproducible VNTR repeat counts as standardized outputs for reliable isolate comparison.
- Analytics: Size calling and in silico repeat count calculation provide measurable readouts for statistical analysis and cluster formation.
- Translational Research: Connects discovery-phase genotyping to preclinical continuity through spatiotemporal strain mapping and infection source tracking.
- Enterprise Reuse: Standardized protocol and portable data format allow reuse across sites for consistent epizootiological surveillance.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in strain discrimination reduces mechanistic ambiguity in pathogen behavior and host interaction studies.
- Operational Value: Boiling-based DNA extraction and multiplex PCR ensure low-cost, scalable, and reproducible sample processing.
- Strategic Value: Enables better go/no-go decisions in vaccine strain selection by identifying dominant virulent clones in specific regions.
- Portfolio Impact: Risk-adjusted prioritization of interventions based on MLVA-defined cluster analysis of field isolates.
Implementation Considerations
- Requires expertise in molecular microbiology and fragment analysis techniques.
- Needs access to PCR thermocycler, gel electrophoresis, and capillary electrophoresis systems.
- Demands standardization of primer concentrations and cycling conditions across two five-plex reactions.
- Requires adaptation of size-calling protocols to account for locus-specific amplicon mobility biases.
- Dependent on bioinformatics tools like BioNumerics and Gene Mapper for data analysis and MST generation.
Biopharma FAQs
Chapters in this video
0:04
Title
0:53
Bacterial Cultivation and Extraction of Genomic DNA
1:54
Multiplex PCR Setup and Cycling Conditions
3:14
PCR Amplicon Confirmation by Gel Electrophoresis
4:37
Capillary Electrophoresis Setup and Run Conditions
6:06
VNTR (Variable-number Tandem-repeat) Size Calling, Repeat Count Calculation and MLVA (Multi-locus Variable-number Tandem-repeat Analysis) Profiling
7:36
Minimum Spanning Tree Cluster Analysis of MLVA Data
9:21
Results: Gel Electrophoresis, Electropherograms, and Minimum Spanning Tree
10:09
Conclusion
