Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of ~200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.
Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.
Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) are two poultry pathogens affecting the respiratory tract of chickens, and cause major economic losses in the industry. Rapid detection of these viruses is crucial to inform implementation of appropriate control measures. The objective of our study is developing a simple, rapid and field applicable recombinase polymerase amplification (RPA)-nucleic acid lateral flow (NALF) immunoassay for detection of NDV and IBV. Isothermal amplification of the matrix protein (M) gene of NDV and the nucleoprotein (N) gene of IBV was implemented via recombinase polymerase amplification at 38 °C for 40 min and 20 min, respectively using modified labeled primers. NALF device used in this study utilizes antibodies for detection of labeled RPA amplicons. The results revealed that RPA-NALF immunoassays can detect both viruses after 40 min at 38 °C and only NDV after 20 min. The limit of detection (LOD) was 10 genomic copies/RPA reaction. The assays results on clinical samples collected from diseased chicken farms demonstrated a good performance in comparison with quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). The assays established in this study can facilitate rapid, on-site molecular diagnosis of suspected cases of ND and IB viral infections as the results can be detected by the naked eye without the need for measuring fluorescence. Furthermore, the NALF device could be adapted to detect other infectious agents.
An artificial intelligence-assisted low-cost portable device for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presented here. This standalone temperature-controlled device houses tubes designed for conducting reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays. Moreover, the device utilises tubes illuminated by LEDs, an in-built camera, and a small onboard computer with automated image acquisition and processing algorithms. This intelligent device significantly reduces the normal assay run time and removes the subjectivity associated with operator interpretation of colourimetric RT-LAMP results. To further improve this device’s usability, a mobile app has been integrated into the system to control the LAMP assay environment and to visually display the assay results by connecting the device to a smartphone via Bluetooth. This study was undertaken using ~5000 images produced from the ~200 LAMP amplification assays using the prototype device. Synthetic RNA and a small panel of positive and negative SARS-CoV-2 patient samples were assayed for this study. State-of-the-art image processing and artificial intelligence algorithms were applied to these images to analyse them and to select the most efficient algorithm. The template matching algorithm for image extraction and MobileNet CNN architecture for classification results provided 98.0% accuracy with an average run time of 20 min to confirm the endpoint result. Two working points were chosen based on the best compromise between sensitivity and specificity. The high sensitivity point has a sensitivity value of 99.12% and specificity value of 70.8%, while at the high specificity point, the sensitivity is 96.05% and specificity 93.59%. Furthermore, this device provides an efficient and cost-effective platform for non-health professionals to detect not only SARS-CoV-2 but also other pathogens in resource-limited laboratories, factories, airports, schools, universities, and homes.
HighlightsA low cost, optical, POC molecular diagnostic platform.Sample preparation using a paper membrane.Isothermal amplification using HDA and RPA.
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