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A novel RNA detection technique for point-of-care identification of pathogens
Mekbib Astatke1, Olivia Tiburzi1, Amy Connolly2
1Applied Biological Sciences, The Johns Hopkins University Applied Physics Laboratory, Laurel, United States.
Abstract:
Despite significant progress in recent years to improve capabilities to diagnose infections at point-of-care (POC), there are still technical hurdles that need to be overcome to ensure proper medical interventions. Current microbial POC tests involve polymerase chain reaction (PCR) or sandwich immunoassay (IA) based detection formats. PCR is highly sensitive but requires complex instrumentation, whereas lateral flow (LF) based IA tests are handheld but lack sensitivity. We present here a portable and sensitive technique by integrating an isothermal RNA amplification approach with IA detection format. The technique comprises i) Nucleic Acid Sequence Based isothermal Amplification (NASBA), ii) amplicon tagging with hapten labeled probes, iii) capturing the amplicon and iv) formation of a sandwich complex with an antibody (Ab) that selectively recognizes the DNA-RNA duplex. The results can be extended to develop an automated, portable and highly sensitive diagnostic platform suitable for POC applications.
Insights
This study introduces a sensitive and portable diagnostic method by combining Nucleic Acid Sequence Based Amplification (NASBA) with immunoassays. This approach enhances point-of-care infection detection capabilities.
Area of Science:
- Biotechnology
- Medical Diagnostics
- Molecular Biology
Background:
- Current point-of-care (POC) infection diagnostics face limitations: Polymerase Chain Reaction (PCR) is sensitive but requires complex equipment, while lateral flow immunoassays (IA) are portable but lack sensitivity.
- Bridging the gap between sensitivity and portability is crucial for effective, timely medical interventions in infectious disease diagnosis.
Purpose of the Study:
- To develop a novel, portable, and highly sensitive diagnostic technique for point-of-care (POC) applications.
- To integrate isothermal RNA amplification with immunoassay detection for improved microbial diagnostics.
Main Methods:
- Nucleic Acid Sequence Based Amplification (NASBA) for isothermal RNA amplification.
- Development of a sandwich immunoassay format involving hapten-labeled probes and specific antibody recognition of DNA-RNA duplexes.
- Integration of amplicon capture and detection into a single workflow.
Main Results:
- Demonstration of a portable technique combining NASBA with IA for sensitive detection.
- Successful tagging of amplicons and formation of a sandwich complex for signal generation.
- Potential for automation and high sensitivity in a POC setting.
Conclusions:
- The integrated NASBA-IA technique offers a promising solution for sensitive and portable infection diagnostics.
- This approach addresses limitations of existing POC methods, paving the way for advanced automated diagnostic platforms.
- The developed method has significant potential for improving infectious disease management at the point-of-care.
