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 often require enrichment cultures and are not suitable for distinguishing between species like C. jejuni, C. coli, and other Campylobacter species, leading to difficulties in identifying C. hyointestinalis.
Innovation Solution
Development of a method targeting the cytolethal distending toxin (CDT) genes using multiplex PCR and specific primers 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 enable species-specific detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
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 specific to these genes, the method directly detects the presence of Campylobacter species through gene amplification, bypassing the lengthy cultivation process while maintaining identification accuracy
Solution Approach 2:
The invention replaces the mechanical/cultural system of bacterial cultivation with a molecular biological system (PCR-based gene detection). Instead of relying on bacterial growth and biochemical property analysis, the method uses nucleic acid amplification and detection to identify Campylobacter species, significantly reducing detection time from 7-10 days to a few hours
2Measurement precision
If PCR methods for detecting hippuricase gene are used to distinguish C. jejuni and C. coli, then detection can be performed, 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 local regions (the cdtA, cdtB, and cdtC genes) within the Campylobacter genome that exhibit species-specific variations. These particular gene regions contain sufficient sequence diversity to differentiate between C. jejuni, C. coli, and other Campylobacter species, while the rest of the genome remains highly homologous
Solution Approach 2:
The invention segments the detection approach by using multiple specific primer pairs, each targeting a different gene (cdtA, cdtB, or cdtC). This segmentation allows comprehensive differentiation of Campylobacter species by analyzing multiple genetic markers simultaneously, increasing both accuracy and reliability of species identification
3Productivity
If selection media and culture conditions developed for C. jejuni and C. coli are used, then isolation of these species can be achieved, but bacterial species other than C. jejuni and C. coli cannot be isolated due to differences in antibiotic sensitivity or optimal culture temperature
Solution Approach 1:
The invention achieves universality by developing a PCR detection system that can identify multiple Campylobacter species (C. jejuni, C. coli, C. fetus, and others) using a common methodological framework. By designing primers that target conserved regions of the cdt genes present across different species, the method provides broad coverage while maintaining species-specific differentiation capability
Solution Approach 2:
The invention applies dynamics by making the detection system adaptable to different Campylobacter species through the use of multiple specific primer pairs. The system can be dynamically configured by selecting appropriate primer pairs based on the target species, allowing flexible and versatile detection across the entire Campylobacter genus without being restricted to fixed culture conditions
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
This approach allows for rapid and accurate detection of Campylobacter hyointestinalis and other species, overcoming the limitations of existing methods by providing specific primers and probes that can differentiate between C. jejuni, C. coli, and C. hyointestinalis, facilitating the identification of Campylobacter species that cause food poisoning.
Implementation Method 1
PCR methods for detecting the presence of the hippuricase gene have been used in actual tests
Implementation Method 2
16S rRNA gene analysis is frequently used as a method for identifying bacterial species at the gene level
Data Source
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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.