Visual LAMP Method for Rapid Tobacco Identification
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Solution Overview
Problem
Current molecular biological methods for tobacco identification, such as SCAR, RAPD, AFLP, SSR, and SNP, require complex devices like PCR and genome sequencing, limiting their application in frontline tobacco product inspection.
Innovation Solution
A visual loop-mediated isothermal amplification (LAMP) method is developed for rapid tobacco testing, involving genomic DNA extraction using Chelex-100, primer design, and optimization of the LAMP reaction system with SYBR Green I for color determination, allowing for rapid and sensitive identification of tobacco species.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional molecular biological methods (SCAR, RAPD, AFLP, SSR, SNP) are used for tobacco identification, then identification accuracy is improved, but device complexity and operational difficulty increase
Solution Approach 1:
The patent changes the reaction parameters from complex multi-step PCR cycles to simple isothermal amplification at constant temperature (65°C). This parameter change simplifies the device requirements while maintaining identification accuracy through the use of specific LAMP primers and SYBR Green I staining for visual detection
Solution Approach 2:
The patent replaces complex mechanical PCR cycling systems with a simple thermal incubation system operating at constant temperature. The LAMP method substitutes the need for complex temperature cycling equipment with basic heating blocks or water baths, significantly reducing device complexity while achieving the same identification function
2Measurement precision
If traditional molecular biological methods are used for tobacco identification, then identification accuracy is improved, but ease of operation deteriorates
Solution Approach 1:
The LAMP method enables frontline inspectors to perform autonomous tobacco identification without requiring specialized molecular biology expertise or complex equipment operation. The visual color change provides self-indicating results that can be interpreted by non-experts, making the method self-service friendly for field inspection personnel
Solution Approach 2:
The patent simplifies operational parameters by using isothermal amplification instead of complex PCR cycling programs. The constant temperature operation (65°C) and visual colorimetric detection eliminate the need for sophisticated instrument operation, making the method easily operable by frontline inspection personnel with minimal training
3Productivity
If rapid DNA extraction method is used, then testing speed is improved, but extraction completeness may worsen
Solution Approach 1:
The patent uses Chelex-100 resin as an intermediary agent in the rapid DNA extraction process. This chelating resin binds to metal ions and facilitates rapid cell lysis and DNA release without requiring complex purification steps. The extracted DNA is sufficient for LAMP amplification, achieving both speed and adequate completeness for detection purposes
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 method enables rapid DNA extraction and LAMP testing within 20-45 minutes, achieving a detection limit of 200 copies per reaction with high specificity and a 96.7% coincidence rate with qPCR, facilitating quick inspection of tobacco products.
Implementation Method 1
a LAMP amplification product is mixed with a color-producing cap including SYBR Green I; and a color of a reaction solution is observed to obtain a determination result
Implementation Method 2
carrying out a LAMP reaction at 63°C
Implementation Method 3
centrifuging for 2 min at 12,000 r/min, and taking a supernatant for testing
Implementation Method 4
adding 1/10 volume of proteinase K and 1/10 volume of RnaseA, shaking and suspending for 10-30 s, and water-bathing at 55° C. for 5 min
Implementation Method 5
boiling at 100° C. for 5 min
Data Source
AI summary
A visual loop-mediated isothermal amplification (LAMP) method for a rapid test of tobacco includes: extracting a genomic DNA of the tobacco, designing primers, establishing a LAMP reaction system, and optimizing the LAMP reaction system. The present disclosure designs the LAMP primers according to a conserved region of a screened tobacco-specific Ntsp151 genomic sequence, and establishes the LAMP method based on color determination. The present disclosure can rapidly identify flue-cured tobacco leaves, finished cigarettes, and tea cigarettes, and achieves a detection limit of 200 copies per reaction. The test results of the LAMP method for non-tobacco samples are negative, indicating that the LAMP method has high specificity. Compared with the quantitative polymerase chain reaction (qPCR) method, the LAMP method has a coincidence rate of 96.7%, but the LAMP method only needs about 1 h to obtain the results.