Opportunity
Current pathogen detection methods, particularly those based on loop-mediated isothermal amplification (LAMP), face significant challenges in balancing speed, specificity, and ease of use. Traditional LAMP assays often suffer from false-positive results due to non-specific amplification or signal errors (e.g., pH fluctuations in samples). Additionally, conventional LAMP requires a fixed incubation time to ensure complete amplification, delaying results. Immunoassays like lateral flow assays (LFA) offer high specificity but are typically endpoint tests, requiring prior completion of amplification. The lack of integration between real-time LAMP (e.g., colorimetric/fluorescence) and LFA limits the ability to achieve rapid, high-specificity diagnostics, especially in resource-limited settings. This patent addresses these gaps by combining real-time LAMP with LFA in a single reaction system, enabling early termination of amplification upon positive signals and validation via LFA.
Technology
The patent introduces a one-pot pathogen detection system that integrates LAMP with real-time colorimetric/fluorescence readouts and LFA. Key innovations include:
- Dual-Indicator System: The reaction mixture contains a colorimetric indicator (e.g., phenol red, hydroxy naphthol blue) and a fluorescent dye (e.g., SYBR Green) to monitor amplification in real time via pH/metal ion changes or fluorescence spikes.
- Primer Tagging: Select LAMP primers (e.g., FIP, BIP, LB) are labeled with two distinct haptens (e.g., biotin, FAM) to enable LFA detection of amplification products. This ensures high specificity by requiring dual hapten-antibody binding for a positive result.
- Early Termination: The reaction stops when color/fluorescence signals indicate amplification, followed by immediate LFA validation, reducing false positives from prolonged incubation.
- Versatile Platform: The system works on simple heating devices (e.g., water baths, heating blocks), eliminating the need for complex instrumentation.
Advantages
- Faster Results: Reduces LAMP reaction time by up to 50% by terminating upon early positive signals.
- High Specificity: LFA confirmation minimizes false positives from non-specific amplification.
- Multimodal Detection: Combines colorimetric, fluorescent, and LFA readouts for robust result interpretation.
- Portability: Compatible with low-resource settings due to minimal equipment requirements.
- Broad Applicability: Detects DNA/RNA from viruses (e.g., MPXV, SARS-CoV-2), bacteria, and fungi.
Applications
- Point-of-Care Diagnostics: Rapid detection of pathogens like MPXV, SARS-CoV-2 in field or clinical settings.
- Disease Surveillance: High-throughput screening during outbreaks.
- Veterinary and Agricultural Use: Detection of animal or plant pathogens.
- Research Tools: Multiplexed pathogen detection in lab environments.
