ABSTRACT
Introduction
Suitable sample collection and preparation methods are essential to enable nucleic acid amplification testing at the point of care (POC). Strategies that allow direct isothermal nucleic acid amplification testing (iNAAT) of crude sample lysate without the need for nucleic acid extraction minimize time to result as well as the need for operator expertise and costly infrastructure.
Areas covered
The authors review research to understand how sample matrix and preparation affect the design and performance of POC iNAATs. They focus on approaches where samples are directly combined with liquid reagents for preparation and amplification via iNAAT strategies. They review factors related to the type and method of sample collection, storage buffers, and lysis strategies. Finally, they discuss RNA targets and relevant regulatory considerations.
Expert opinion
Limitations in sample preparation methods are a significant technical barrier preventing implementation of nucleic acid testing at the POC. The authors propose a framework for co-designing sample preparation and amplification steps for optimal performance with an extraction-free paradigm by considering a sample matrix and lytic strategy prior to an amplification assay and readout. In the next 5 years, the authors anticipate increasing priority on the co-design of sample preparation and iNAATs.
Article highlights
Point of care isothermal nucleic acid amplification tests (iNAATs) require effective sample preparation workflows that can also be implemented at the POC.
Workflows that are extraction-free – excluding isolation, concentration, or purification of nucleic acids can minimize requirements of infrastructure, personnel, and time.
Choosing a matrix with a high concentration of target, designing a compatible preparation strategy, and designing a readout strategy to maximize likelihood of compatibility enable assay performance.
Following strategic design, increasing co-factor concentration, buffering pH, employing nucleic acid protectors, and adjusting reaction viscosity provide further opportunities for integrated assay optimization.
Future work should treat sample preparation as an integral aspect of assay design equal in importance to amplification assay design.
Acknowledgments
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank C Noxon of Rice360 for their technical writing assistance.
Declaration of interest
The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.