Development of a low-temperature nucleic acid amplification assay for the detection of infectious diseases

Background: The polymerase chain reaction (PCR) is a cornerstone of modern molecular biology, enabling on-demand DNA replication and the identification of disease-causing agents. However, its instrumentation requirements limit field deployment. To address this, isothermal nucleic acid amplification (INAA) methods such as Loop-mediated Isothermal Amplification (LAMP) have emerged as promising alternatives for the molecular diagnosis of infectious disease. By employing strand-displacing polymerases that negate the need for thermal cycling, these methods offer reduced costs, faster turnaround, and simpler protocols. While INAA methods show potential for meeting the WHO's ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to end-users) for point-of-care testing, most still require constant incubation at approximately 65 °C and rely on complex readout methods, which prevent truly equipment-free operation and limit their suitability for community healthcare settings. Objective: To develop a nucleic acid amplification assay operating at 37 °C, compatible with both DNA and RNA targets and amenable to simple, equipment-free result interpretation. Methods: The method exploits LNA-modified primers to enable efficient strand invasion at 37 °C. As a proof of concept, primer sets were designed to detect influenza A and influenza B target sequences, and the analytical performance of the assay was characterised, with both real-time and end-point detection achieved by adding DNA-binding dyes such as SYTO 9 and SYBR Green I. Results: The assay achieved a sensitivity of 10³ copies within 60 minutes, approaching the analytical performance of laboratory-based PCR while operating at physiological temperature. Crucially, the low-temperature operation enables several equipment-free design strategies, including incubation using body heat or the use of chemical, electrical, or phase-change materials within a simple device to maintain temperature stability. These features position the assay as a strong candidate for ASSURED-compliant point-of-care testing in community healthcare settings.