ABSTRACT
While the research field and industrial market of in vitro diagnosis (IVD) thrived during and post the COVID-19 pandemic, the development of isothermal nucleic acid amplification test (INAAT) based rapid diagnosis was engendered in a global wised large measure as a problem-solving exercise. This review systematically analyzed the recent advances of INAAT strategies with practical case for the real-world scenario virus detection applications. With the qualities that make INAAT systems useful for making diagnosis relevant decisions, the key performance indicators and the cost-effectiveness of enzyme-assisted methods and enzyme-free methods were compared. The modularity of nucleic acid amplification reactions that can lead to thresholding signal amplifications using INAAT reagents and their methodology design were examined, alongside the potential application with rapid test platform/device integration. Given that clinical practitioners are, by and large, unaware of many the isothermal nucleic acid test advances. This review could bridge the arcane research field of different INAAT systems and signal output modalities with end-users in clinic when choosing suitable test kits and/or methods for rapid virus detection.
ABSTRACT
Automated sample-to-answer systems that promptly diagnose emerging infectious diseases, such as zoonotic diseases, are crucial to preventing the spread of infectious diseases and future global pandemics. However, automated, rapid, and sensitive diagnostic testing without professionals and sample capacity and type limitations remains unmet needs. Here, we developed an automated sample-to-answer diagnostic system for rapid and accurate detection of emerging infectious diseases from clinical specimens. This integrated system consists of a microfluidic platform for sample preparation and a bio-optical sensor for nucleic acid (NA) amplification/detection. The microfluidic platform concentrates pathogens and NAs in a large sample volume using adipic acid dihydrazide and a low-cost disposable chip. The bio-optical sensor allows label-free, isothermal one-step NA amplification/detection using a ball-lensed optical fiber-based silicon micro-ring resonator sensor. The system is integrated with software to automate testing and perform analysis rapidly and simply;it can distinguish infection status within 80 min. The detection limit of the system (0.96 × 101 PFU) is 10 times more sensitive than conventional methods (0.96 × 102 PFU). Furthermore, we validated the clinical utility of this automated system in various clinical specimens from emerging infectious diseases, including 20 plasma samples for Q fever and 13 (11 nasopharyngeal swabs and 2 saliva) samples for COVID-19. The system showed 100% sensitivity and specificity for detecting 33 samples of emerging infectious diseases, such as Q fever, other febrile diseases, COVID-19, human coronavirus OC43, influenza A, and respiratory syncytial virus A. Therefore, we envision that this automated sample-to-answer diagnostic system will show high potential for diagnosing emerging infectious diseases in various clinical applications. © 2023 Elsevier B.V.
ABSTRACT
The demand for scalable, rapid and sensitive COVID-19 diagnostics is particularly pressing at present to help contain the spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. We report the development of an integrated modular centrifugal microfluidic platform costing less than 250 USD to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The platform was validated with a panel of 131 nasopharyngeal swab samples collected from symptomatic COVID-19 patients. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
The SARS-CoV-2 pandemic has elevated the development of novel diagnostic solutions, including rapid nucleic acid amplification tests (NAATs), to a global priority to meet the high demand for accurate, timely viral detection and diagnosis. However, ubiquitously implemented NAATs, such as polymerase chain reaction (PCR), consume hours of testing. We report a field-forward instrument capable of ultra-fast real-time PCR for amplification-based nucleic acid detection in a custom-designed microfluidic chip. Prudent selection and unconventional positioning of thermal cyclers relative to the microfluidic chip and a fluorescent detector permit ultra-fast simultaneous amplification and detection, with 40 cycles complete in under 10 minutes. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
Without global mass vaccination, COVID-19 will continue to infect and cause serious illness, disproportionately in low- and middle-income countries. Point-of-care and home-based nucleic acid amplification tests (NAATs) are valuable tools to control COVID-19 transmission. Here we present a rapid isothermal NAAT for duplexed detection of SARS-CoV-2 and an MS2 bacteriophage internal control. This assay amplifies RNA in less than 15 minutes, utilizes a low temperature of 39°C, and has fluorescence or visual lateral flow readout. This positions our assay for use in low-cost paper-based nucleic acid diagnostic devices for ultrasensitive and reliable COVID-19 detection in POC or home-based settings. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
Nucleic acid amplification detection is one of the most widely used molecular diagnostic techniques in recent years, which can rapidly and efficiently amplify the characteristic nucleotide sequences of pathogenic bacteria in the diagnosis of infectious diseases, it has been widely used in clinical diagnosis, disease screening and other fields. In this work, we report a micro-cavity digital PCR for rapid detection of pathogens on a silicon-based microfluidic chip. The device has the advantages of high flux, no pumping, rapid reaction, quantification and high sensitivity. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
The COVID-19 pandemic demands fast, sensitive, and specific diagnostic tools for virus surveillance and containment. Current methods for diagnosing the COVID-19 are based on direct detection of either viral antigens or viral ribonucleic acids (RNA) in swab samples. Antigen-targeting tests are simple, have fast turnaround times, and allow rapid testing. Unfortunately, compared with viral RNA-targeting tests, their sensitivity is low, especially during the initial stages of the disease, which limits their adoption and implementation. Direct detection of SARS-CoV-2 RNA using reversetranscription quantitative polymerase chain reaction (RT-qPCR) is sensitive and specific, making it a golden standard in SARS-CoV-2 detection. However, it had not seen a significant update since its introduction three decades ago. It has a long turnaround time, requires a high number of amplification cycles, and a complicated and expensive detection system for real-time monitoring of the signal. While insignificant for research applications, these limitations present severe problems for mass testing required to contain the disease. Here, we introduce a diagnostic platform for rapid and highly sensitive clinical diagnosis of COVID-19. Based on the biochemical principles of the RT-PCR, it utilizes the endpoint detection by the magnetic modulation biosensing (MMB) system, allowing the detection of as little as two copies of SARS-CoV-2 in ∼30 minutes. Testing 309 RNA samples from verified SARS-CoV-2 carriers and healthy subjects resulted in 97.8% sensitivity, 100% specificity, and 0% crossreactivity. This level of performance is on par with the gold standard (RT-qPCR) but requires 1/3 of the time. The platform can be easily adapted to detect almost any other pathogen of choice. © COPYRIGHT SPIE. Downloading of the is permitted for personal use only.
ABSTRACT
Loop-mediated isothermal amplification (LAMP) is an exponential amplification method of DNA strands that is more and more used for its high performances. Thanks to its high sensitivity and selectivity, LAMP found numerous applications from the detection of pathogens or viruses through their genome amplification to its incorporation as an amplification strategy in protein or miRNA biomarker quantification. The LAMP method is composed of two stages: the first one consists in the transformation of the DNA strands into dumbbell structures formed of two stems and loops thanks to four primers; then, in the second stage, only two primers are required to amplify the dumbbells exponentially in numerous hairpins of increasing lengths. In this paper, we propose a theoretical framework to analyze the kinetics of the second stage of LAMP, the isothermal dumbbell exponential amplification (IDEA) as function of the physico-chemical parameters of the amplification reaction. Dedicated experiments validate the models. We believe these results may help the optimization of LAMP performances by reducing the number of experiments necessary to find the best parameters.