Campylobacter Detection via cdt Gene Multiplex PCR
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Solution Overview
Problem
Current methods for detecting Campylobacter bacteria, particularly Campylobacter hyointestinalis, are inadequate due to the lack of rapid and specific diagnostic techniques, as existing methods require enrichment cultures and are not effective for distinguishing between species like C. jejuni, C. coli, and C. hyointestinalis.
Innovation Solution
Development of a method using multiplex PCR and specific primers targeting the cdt genes to detect Campylobacter species, including C. hyointestinalis, by amplifying the cdtA, cdtB, and cdtC genes, and determining the entire nucleotide sequence of the C. hyointestinalis cdt genes to create species-specific detection protocols.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cultivation test is used to identify Campylobacter bacterial species, then identification can be performed, but the test requires complex and substantial effort and takes a long time (seven to ten days)
Solution Approach 1:
The invention extracts and targets specific genetic elements (cdtA, cdtB, cdtC genes encoding cytolethal distending toxin) from the complex bacterial identification process. By designing PCR primers that specifically amplify these toxin genes, the method bypasses the need for lengthy cultivation and biochemical testing, achieving rapid identification within hours while maintaining species-level accuracy.
Solution Approach 2:
The invention replaces the mechanical/cultural system of bacterial cultivation with a molecular biological system (PCR amplification and sequencing). Instead of growing bacteria and observing biochemical properties over days, the method directly amplifies and analyzes specific DNA sequences, substituting a faster molecular mechanism for the slower cultural method.
2Productivity
If PCR methods targeting hippuricase gene are used to distinguish C. jejuni and C. coli, then detection speed is improved, but C. jejuni and C. coli are highly homologous and often cannot be distinguished
Solution Approach 1:
The invention applies local quality by targeting specific regions within the cdt genes that exhibit species-specific variations. Rather than using general Campylobacter markers, the method designs primers and probes that focus on localized sequences in the cdtA, cdtB, and cdtC genes where nucleotide differences enable precise discrimination between C. jejuni, C. coli, and other species, maintaining both speed and accuracy.
Solution Approach 2:
The invention segments the identification process into multiple independent PCR reactions, each targeting a different cdt gene (cdtA, cdtB, or cdtC). This segmentation allows comprehensive analysis of species-specific genetic markers across multiple gene regions, improving discrimination accuracy compared to relying on a single gene target.
3Productivity
If selection media and culture conditions are optimized for C. jejuni and C. coli, then isolation of these species is improved, but other Campylobacter species cannot be isolated due to differences in antibiotic sensitivity or optimal culture temperature
Solution Approach 1:
The invention creates a universal detection system that can identify multiple Campylobacter species simultaneously. By designing PCR primers that target conserved regions of the cdt genes present across different species, the method achieves multi-functionality, enabling detection of C. jejuni, C. coli, C. fetus, and other species in a single assay platform, eliminating the need for species-specific optimization.
4Loss of time
If rapid identification methods are developed, then detection time is reduced, but existing methods lack specificity for Campylobacter hyointestinalis and other less common species
Solution Approach 1:
The invention changes the detection parameters by targeting the cdt genes, which show sufficient sequence divergence among Campylobacter species. By adjusting the primer sequences and hybridization conditions to match species-specific variations in the cdtA, cdtB, and cdtC genes, the method achieves both rapid detection and high specificity for C. hyointestinalis and other less common species that were previously undetectable.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables rapid and specific detection of Campylobacter hyointestinalis and other species, improving diagnostic capabilities and overcoming the limitations of existing methods by providing a novel approach for identifying Campylobacter bacteria beyond C. jejuni and C. coli.
Implementation Method 1
PCR methods for detecting the presence of the hippuricase gene have been used in actual tests
Implementation Method 2
The CdtB subunit has DNase activity and is the main unit of toxin that exerts toxicity
Data Source
AI summary
An objective of the present invention is to provide the cytolethal distending toxin (CDT) of C. hyointestinalis and polynucleotides encoding it, and novel methods for detection of C. hyointestinalis using the cdt genes. The present inventors focused on the cytolethal distending toxin (CDT) of Campylobacter bacteria, and detected the cdt genes of a Campylobacter-like bacterium isolated from an enteritis patient in Thailand. The present inventors discovered a bacterial strain whose cdtB gene was amplified by common primers in C. jejuni, C. coli, and C. fetus, but not by multiplex PCR that can specifically detect the cdtA, cdtB, and cdtC genes of the three bacterial species. The bacterial strain was identified as C. hyointestinalis by 16S rRNA gene analysis. Furthermore, the entire nucleotide sequence of the cdt genes was determined by genome walking upstream and downstream of the cdtB gene.