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Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care
Lucas Rodrigo de Souza1,2, Italo Esposti Poly da Silva1,2,3, Gabriele Celis-Silva1,2
1Center for Molecular Biology and Genetic Engineering (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas/SP 13083-875, Brazil.
Experimental Biology and Medicine (Maywood, N.J.)
|December 13, 2023
Summary
Researchers optimized enzymes for reverse transcription loop-mediated isothermal amplification (RT-LAMP), enhancing diagnostic capabilities. These stable, high-purity enzymes are crucial for sensitive detection of viral genetic material in point-of-care settings.
Area of Science:
- Molecular Diagnostics
- Biotechnology
- Infectious Disease Preparedness
Background:
- The COVID-19 pandemic highlighted the need for rapid and scalable diagnostic methods for public health emergencies.
- Traditional methods like RT-qPCR face limitations in resource-limited settings.
- Reverse transcription loop-mediated isothermal amplification (RT-LAMP) emerged as a promising alternative for sensitive nucleic acid detection.
Purpose of the Study:
- To improve the production and stability of key enzymes for RT-LAMP assays.
- To optimize enzyme purification for increased efficiency and preserved activity.
- To validate the performance of the optimized RT-LAMP system in a point-of-care format.
Main Methods:
- Optimized protein production protocols for *Bst* polymerase and *HIV*-reverse transcriptase.
- Evaluated different protein tags and streamlined purification procedures.
- Assessed enzyme stability at various temperatures (4°C and 25°C) over 60 days.
- Tested the one-step RT-LAMP reaction using clinical samples on a portable device.
Main Results:
- Achieved high purity and preserved activity of RT-LAMP enzymes through optimized production and purification.
- Demonstrated significant enzyme stability for over 60 days at common storage temperatures.
- Confirmed the reliability of the one-step RT-LAMP reaction with clinical samples in a point-of-care device.
Conclusions:
- Enhanced production and purification strategies yield stable and active RT-LAMP enzymes.
- The optimized RT-LAMP system offers a sensitive and reliable diagnostic tool for point-of-care applications.
- This approach has potential for broader application in detecting various infectious agents.
