Portable molecular diagnostics by convective PCR and multiplex detection by smartphone reader
The widespread implementation of nucleic acid-based diagnostics in low-resource settings requires portable and readily accessible technologies which provide quantitative information without the need for sophisticated instruments and highly-trained technicians. A promising solution for rapid and simple nucleic acid amplification is the use of Rayleigh– Bernard natural convection, which is caused by buoyancy-driven thermal gradients of liquid when heated from below using a single heater at a constant temperature. Natural convection avoids the use of complex and sophisticated heaters and ramping that is required for precise maintenance of temperature cycles in the conventional polymerase chain reaction (PCR). Convective flow can transport reagents continuously through the specific temperature regions required for each step of PCR and perform 35 - 40 cycles in less than 30 minutes. This ability drastically reduces the electrical power requirement, cost and the time required for nucleic acid amplification.
A standalone convective PCR (cPCR) device was developed for rapid pseudo-isothermal amplification of nucleic acid using natural convection. The device can amplify multiple samples simultaneously using a custom-made single heat block controlled by Bluetooth communication through a dedicated smartphone application developed for this purpose. The entire device is highly portable, user-friendly and battery operated. The amplicons obtained by cPCR were detected and quantified using two different approaches. First, fluorescenttagged cPCR amplicons were concentrated magnetically, enriched in a PCR tube and detected on a smartphone fluorescence reader. As an alternative approach, amplicons were detected using a nucleic acid lateral flow immunoassay to provide specific detection on a nitrocellulose membrane in few minutes. In both approaches, a customised fluorescence tube/strip reader was developed and attached to the smartphone for quantitative analysis. Using these procedures, detection limits of 2.8 x 103 copies of lambda DNA and 4.7 x 103 copies of methicillin resistant Staphylococcus aureus genomic DNA were obtained. To reduce the read-out complexity, the captured images were analysed on the smartphone using the Android free image analysis application, IJ_Mobile. Image analysis was displayed on the smartphone as a graphical read-out, allowing the results to be archived and/or easily transmitted from remote locations.
One of the inherent advantages of molecular diagnostics is the ability to multiplex and detect multiple targets simultaneously and to facilitate inclusion of appropriate internal controls. The detection strategies described above were multiplexed using various combinations of fluorophores/quantum dots to allow for simultaneous detection of antibiotic resistance genes, as a proxy for enabling rapid detection of infectious pathogens. The performance of the cPCR assay and integrated detection system was analysed successfully for sensitivity, reproducibility and portability using methicillin-resistant S. aureus and lambda DNA as target samples. The results suggested that the simple design and user-friendly nature of the smartphone-integrated cPCR is well-suited for on-site molecular diagnostics in remote and/or low-resource settings. The technology has significant potential in contributing to more rapid and sensitive results without the necessity for expensive and specialised equipment, thus allowing for better choice of intervention, improved clinical practices and more reliable decision-making.