Lactobacillus casei Identification via mutL dnaJ Degenerate Primers
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Solution Overview
Problem
Current methods for distinguishing between species and strains of Lactobacillus casei group bacteria, such as PFGE and MLST, are complex, time-consuming, and lack reproducibility, making it difficult to accurately identify inter- and intraspecies relationships.
Innovation Solution
A degenerate primer pair is used to amplify partial fragments of the mutL and dnaJ genes, allowing for sequencing and comparison to determine species and strain-level identification through high similarity and identity analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PFGE is used for species discrimination, then intraspecies discrimination is achieved, but the method becomes complex and time-consuming
Solution Approach 1:
The patent extracts and sequences specific gene regions (mutL, dnaJ, recA) from the bacterial genome rather than performing whole-genome PFGE analysis. By focusing on particular genetic markers, the method simplifies the discrimination process while maintaining species and strain identification accuracy.
Solution Approach 2:
The patent replaces the mechanical PFGE separation process with molecular sequencing and bioinformatic analysis. Instead of physically separating bacterial genomes based on restriction enzyme cuts, the method uses DNA sequencing to compare genetic sequences, thereby reducing operational complexity.
2Reliability
If MLST is used for taxonomic classification, then good discrimination and reproducibility are achieved, but the process becomes time-consuming and costly
Solution Approach 1:
The patent extracts and sequences specific gene regions (mutL, dnaJ, recA) from the bacterial genome rather than performing whole-genome PFGE analysis. By focusing on particular genetic markers, the method simplifies the discrimination process while maintaining species and strain identification accuracy.
Solution Approach 2:
The patent sequences only the necessary portions of the genome (specific housekeeping genes) rather than performing complete genomic analysis. This partial sequencing approach provides sufficient discrimination power while significantly reducing time and resource requirements compared to full MLST protocols.
3Measurement precision
If multiple gene loci are sequenced for MLST, then discrimination accuracy is improved, but the cost and time required increase
Solution Approach 1:
The patent focuses on sequencing specific local regions of the genome (mutL, dnaJ, recA genes) that contain sufficient variability for discrimination. By concentrating on these locally important genetic markers rather than sequencing entire genomes or multiple gene loci, the method achieves accurate discrimination with reduced sequencing volume.
Solution Approach 2:
The patent extracts and sequences specific gene regions (mutL, dnaJ, recA) from the bacterial genome rather than performing whole-genome PFGE analysis. By focusing on particular genetic markers, the method simplifies the discrimination process while maintaining species and strain identification accuracy.
Data Source
AI summary
Degenerate primers for amplifying fragments of the nucleotide sequences of mutL and dnaJ genes of a lactic acid bacterium of Lactobacillus casei group are provided. Methods and kits for discriminating interspecies and intraspecies of a lactic acid bacteria of Lb. casei group are also provided.