Fusobacterium nucleatum Detection via LAMP Primer Specificity
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Solution Overview
Problem
Current methods for detecting Fusobacterium nucleatum and its subspecies lack specificity and sensitivity, particularly in distinguishing between the four subspecies without strain-level identification.
Innovation Solution
A method involving loop-mediated isothermal amplification (LAMP) using primer sets specific to the citrate lyase beta subunit gene and the dicarboxylate/amino acid:cation symporter gene for the detection of F. nucleatum and its subspecies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If 16S rRNA probe method is used to detect F. nucleatum, then detection can be performed, but specificity deteriorates due to cross-reaction with various bacteria including Campylobacter
Solution Approach 1:
The patent applies local quality by designing primers that target a specific local region (citrate lyase beta subunit gene) within the F. nucleatum genome rather than using a general 16S rRNA probe. This localized targeting ensures high specificity to F. nucleatum while avoiding cross-reactions with other bacteria.
Solution Approach 2:
The patent segments the detection approach by dividing the genome into specific functional regions and selecting the citrate lyase beta subunit gene as the target. This segmentation allows for precise targeting of F. nucleatum-specific sequences rather than relying on conserved regions that cross-react with other bacteria.
2Measurement precision
If CRISPR-Cas sequence detection method is used to identify F. nucleatum at strain level, then identification precision improves, but device complexity and ease of operation deteriorate due to requiring electrophoresis or nucleotide sequence determination
Solution Approach 1:
The patent replaces complex mechanical systems (electrophoresis apparatus, sequencing equipment) with a simpler nucleic acid amplification system using PCR or LAMP. The method substitutes physical separation and sequencing processes with targeted amplification and detection of specific gene regions, dramatically simplifying the detection workflow while maintaining strain-level identification precision.
Solution Approach 2:
The patent changes the detection parameter from requiring full sequence determination to detecting specific amplification products of the citrate lyase beta subunit gene. By changing the parameter from comprehensive sequencing to targeted amplification detection, the method achieves high precision identification with simpler equipment and procedures.
3Measurement precision
If CRISPR-Cas sequence detection method is used to identify F. nucleatum at strain level, then identification precision improves, but productivity deteriorates due to complex procedures
Solution Approach 1:
The patent replaces time-consuming mechanical procedures (electrophoresis, sequencing) with a rapid nucleic acid amplification system. The PCR or LAMP method amplifies the target gene region exponentially in a single tube, eliminating multiple processing steps and significantly improving detection throughput and productivity while maintaining high identification precision.
4Adaptability or versatility
If no species-specific sequence is found other than 16S rRNA gene and CRISPR, then detection versatility is limited, but manufacturing precision improves by using common genes
Solution Approach 1:
The patent changes the parameter from using highly conserved common genes (16S rRNA, CRISPR) to using a species-specific functional gene (citrate lyase beta subunit gene). This parameter change enables the development of primers with high sequence specificity to F. nucleatum, allowing versatile and specific detection across different F. nucleatum strains while avoiding cross-reactions.
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 method enables specific and sensitive detection of F. nucleatum and its subspecies, including F. nucleatum subsp. nucleatum, in clinical specimens without the need for culture, improving diagnostic accuracy and efficiency.
Implementation Method 1
a step of performing a nucleic acid amplification reaction on a DNA derived from the sample using a primer set specific to a citrate lyase beta subunit gene of Fusobacterium nucleatum
Implementation Method 2
the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction
Data Source
AI summary
According to one aspect of the present invention, this method for detecting Fusobacterium nucleatum in a sample involves a step for subjecting a DNA derived from a sample to nucleic acid amplification reaction using a primer set specific to a citrate lyase beta subunit gene of Fusobacterium nucleatum. According to said method, F. nucleatum can be specifically detected. According to another aspect of the present invention, this method for detecting Fusobacterium nucleatum nucleatum in a sample involves a step for subjecting a DNA derived from a sample to nucleic acid amplification reaction using a primer set specific to a dicarboxylate/amino acid: cation cotransporter gene of Fusobacterium nucleatum nucleatum. According to said method, F. nucleatum nucleatum can be specifically detected.