Mycobacterium DNA Primer Pair for Rapid, Specific PCR Detection
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Solution Overview
Problem
Current methods for detecting Mycobacterium DNA lack sensitivity, specificity, and standardization, making them inadequate for rapid and reliable detection in biological products, particularly in the context of biologics production and vaccine development.
Innovation Solution
A primer pair and detection reagent are developed, comprising specific forward and reverse primers that bind to Mycobacterium genomic DNA sequences, along with a probe, enabling PCR-based detection with high sensitivity and specificity, capable of distinguishing Mycobacterium from interfering DNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional culture methods or guinea pig inoculation are used for Mycobacterium detection, then the detection can be performed with simple equipment and procedures, but the detection time is extremely long (up to 56 days for culture, 4 weeks observation plus tuberculin test for guinea pig method) and sensitivity is insufficient
Solution Approach 1:
The patent replaces traditional mechanical/biological detection systems (culture methods requiring incubation, guinea pig inoculation requiring observation periods) with a molecular biology system based on PCR amplification. The specific primer pairs (SEQ ID NO: 1-6) enable direct DNA detection, reducing detection time from weeks to hours while maintaining high sensitivity through specific amplification of Mycobacterium genomic DNA sequences.
Solution Approach 2:
The patent changes the detection parameter from observing phenotypic characteristics (colonies, nodules) to detecting genotypic characteristics (specific DNA sequences). By designing primers that bind to unique Mycobacterium DNA regions, the method achieves rapid identification without requiring long-term culture or animal observation, fundamentally changing the detection timescale and sensitivity parameters.
2Reliability
If PCR method is used to improve detection sensitivity and speed, then detection time is reduced and sensitivity is improved, but the method lacks standardization and specificity for distinguishing Mycobacterium from interfering DNAs
Solution Approach 1:
The patent segments the detection process into multiple specific steps: DNA extraction, PCR amplification with specific primer pairs, and probe hybridization. Each step is optimized for Mycobacterium detection, with primers designed to bind to unique genomic regions, ensuring high specificity. The segmented approach allows rigorous validation of each step's contribution to overall precision.
Solution Approach 2:
The patent creates a universal detection system that can identify multiple Mycobacterium species through conserved genomic regions while maintaining species-specific discrimination. The primer pairs are designed to target universal Mycobacterium sequences, enabling the same methodology to detect diverse species (M. avium, M. tuberculosis, M. bovis, etc.) with equal specificity, making the system broadly applicable across different biological products.
3Adaptability or versatility
If the scope of mycobacterial testing is expanded to cover all 200+ Mycobacterium species, then the safety coverage is improved, but the complexity of validation and method approval increases significantly
Solution Approach 1:
The patent designs a universal PCR-based detection system that can simultaneously detect multiple Mycobacterium species using a single set of primer pairs. By targeting conserved genomic regions present across all Mycobacterium species, the method achieves broad coverage without requiring separate validated methods for each species, significantly reducing validation complexity while maintaining comprehensive safety coverage.
Solution Approach 2:
The patent changes the approach from species-specific detection (requiring separate methods for each Mycobacterium species) to genus-level detection with species differentiation capability. This parameter change allows a single standardized method to cover all 200+ species, reducing the number of validations needed from hundreds to one, while still providing comprehensive safety coverage through the ability to detect any Mycobacterium contamination.
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
The primer pair achieves high sensitivity, allowing detection of as low as 10 CFU of Mycobacterium, and differentiates Mycobacterium from other bacteria and fungi, ensuring rapid, specific, and standardized detection.
Implementation Method 1
a primer pair for detecting Mycobacterium genomic DNA, comprising a forward primer and a reverse primer, wherein the forward primer binds to positions 510-550... of the sequence shown in SEQ ID NO: 1 on the Mycobacterium genomic DNA; the reverse primer binds to positions 740-780... of the sequence shown in SEQ ID NO: 1 on the Mycobacterium genomic DNA
Implementation Method 2
the length of the product obtained through the amplification by said primer pairs is 240 to 700 bp
Data Source
AI summary
The present invention provides a primer pair for detecting a Mycobacterium genomic DNA, a detection reagent containing the primer pair of the present invention, and a method for detecting the Mycobacterium genomic DNA by using the primer pair, wherein the primer pair specifically binds to a sequence as shown in SEQ ID NO: 1.