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Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification: A Novel Analytically Rapid,
Yi Wang1, Yan Wang1, Ruiting Lan2
1State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
Multiplex detection using loop-mediated isothermal amplification (LAMP) is now possible with a novel endonuclease restriction method. This technique enables rapid, sensitive, and specific identification of multiple DNA targets in a single reaction.
Area of Science:
- Molecular Biology
- Biotechnology
- Diagnostic Assays
Background:
- Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique known for its speed and simplicity.
- However, traditional LAMP is limited to detecting only a single target, restricting its application in complex biological samples.
Purpose of the Study:
- To develop a novel multiplex LAMP assay for simultaneous detection of multiple DNA targets.
- To enhance the sensitivity, specificity, and speed of LAMP-based diagnostics.
Main Methods:
- Developed a multiple endonuclease restriction real-time-LAMP assay utilizing primers with specific recognition sites for the Nb.BsrDI restriction enzyme.
- Primers were modified with a fluorophore and a dark quencher; endonuclease digestion releases quenching, generating a detectable signal.
- Real-time detection of amplified DNA was performed in a single tube.
Main Results:
- The assay successfully achieved real-time, multiplex detection of DNA targets in a single tube.
- Positive results were obtained in as little as 12 minutes.
- Demonstrated high sensitivity (down to 250 fg of DNA) and specificity, with successful application in raw meat sample analysis.
Conclusions:
- The multiple endonuclease restriction real-time-LAMP assay extends LAMP capabilities for robust, target-specific, multiplex detection.
- This novel technology offers a molecular diagnostic tool with reduced detection time, high sensitivity, and specificity compared to conventional LAMP and quantitative real-time PCR.
