LAMP-LFD Primer Kit for Clostridium Piliforme Detection
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Solution Overview
Problem
Current detection methods for Clostridium piliforme, such as reverse dot blot, PCR, and ELISA, are cumbersome, time-consuming, and have low sensitivity and specificity, and involve the use of harmful reagents like calcein fluorescent dye and exposure to carcinogens during electrophoresis, limiting their effectiveness for rapid and accurate detection.
Innovation Solution
A LAMP primer combination including a forward outer primer, backward outer primer, forward inner primer, backward inner primer, loop primer, and probe, combined with LAMP-LFD technology, which uses a kit and method for detecting Clostridium piliforme, allowing for rapid and accurate detection through color changes on chromatographic test strips without the need for precise equipment or harmful reagents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If LAMP amplification is used for detecting Clostridium piliforme, then sensitivity and speed are improved, but harmful factors increase due to use of calcein fluorescent dye and EB carcinogen
Solution Approach 1:
The patent extracts and removes the harmful components (calcein fluorescent dye and EB carcinogen) from the LAMP detection system, replacing them with safe alternatives that do not pose health risks to operators or environmental contamination
Solution Approach 2:
The patent converts the harmful visualization methods into beneficial safe alternatives by using colorimetric changes in the LFD test strip that provide clear detection signals without requiring exposure to carcinogenic dyes or fluorescent markers
2Measurement precision
If traditional detection methods (PCR, ELISA, reverse dot blot) are used, then detection accuracy is achieved, but operation complexity and time consumption increase
Solution Approach 1:
The patent merges the LAMP amplification technology with the LFD detection platform into an integrated one-step system, combining DNA amplification and detection functions to simplify operations while maintaining high detection accuracy
Solution Approach 2:
The LFD test strip serves multiple functions: it detects LAMP amplification products, provides visual colorimetric readout, and eliminates the need for separate electrophoresis or fluorescent detection steps, making the overall system more universal and easier to operate
3Productivity
If LAMP amplification is performed, then detection speed is improved, but harmful factors increase due to carcinogen exposure during electrophoresis
Solution Approach 1:
The patent extracts and eliminates the electrophoresis step that requires EB carcinogen exposure, replacing it with a safe LFD-based detection method that achieves rapid results without harmful chemical exposure
Solution Approach 2:
The LFD test strip acts as an intermediary that directly detects LAMP amplification products without requiring electrophoresis separation, providing a safe bridge between amplification and detection while eliminating carcinogen exposure
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 provides a rapid, accurate, and specific detection of Clostridium piliforme with high sensitivity, 100 times more sensitive than PCR, and eliminates exposure to carcinogens, making it safer and more reliable for on-site detection in experimental animals.
Implementation Method 1
Loop-mediated isothermal amplification (LAMP) technology was used to design four corresponding LAMP primers for six specific sites on a target gene under a certain temperature and the action of a strand displacement Bst 2.0 DNA polymerase
Implementation Method 2
LAMP-amplified products are mainly detected using turbidimetry, electrophoresis, and calcein fluorescent dye. However, LAMP-amplified products are mainly detected using turbidimetry, electrophoresis, and calcein fluorescent dye. During electrophoresis analysis, exposure to the carcinogen EB occurs
Data Source
AI summary
The present disclosure provides a primer combination, a kit, and a method for detecting Clostridium piliforme based on loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD), belonging to the technical field of medical molecular biology detection. In the present disclosure, the primer combination includes a forward outer primer F3, a backward outer primer B3, a forward inner primer FIP, a backward inner primer BIP, a loop primer LF, and a probe PB1. The present disclosure further provides a kit and a method for detecting Clostridium piliforme based on the primer combination. The kit and the method can detect the Clostridium piliforme with a simple detection process, a low time consumption, easy determination of results, and high detection accuracy and sensitivity.