ABSTRACT
Since the COVID-19 pandemic outbreak in the world, many countries have searched for quick diagnostic tools to detect the virus. There are many ways to design diagnostic assays; however, each may have its limitations. A quick, sensitive, specific, and simple approach is essential for highly rapidly transmitted infections, such as SARS-CoV-2. This study aimed to develop a rapid and cost-effective diagnostic tool using a one-step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) approach. The results were observed using the naked eye within 30-60 min using turbidity or colorimetric analysis. The sensitivity, specificity, and lowest limit of detection (LoD) for SARS-CoV-2 RNA against the RT-LAMP assay were assessed. This assay was also verified and validated against commercial quantitative RT-PCR used by health authorities in Saudi Arabia. Furthermore, a quick and direct sampling from the saliva, or buccal cavity, was applied after simple modification, using proteinase K and heating at 98 °C for 5 min to avoid routine RNA extraction. This rapid single-tube diagnostic tool detected COVID-19 with an accuracy rate of 95% for both genes (ORF1a and N) and an LoD for the ORF1a and N genes as 39 and 25 copies/reaction, respectively. It can be potentially used as a high-throughput national screening for different respiratory-based infections within the Middle East region, such as the MERS virus or major zoonotic pathogens such as Mycobacterium paratuberculosis and Brucella spp., particularly in remote and rural areas where lab equipment is limited.
ABSTRACT
We present a nucleic acid-based point-of-care diagnostic for the detection of the SARS-CoV-2 from saliva using an additively manufactured microfluidic cartridge. The assay uses reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) to detect the presence of SARS-CoV-2 RNA on-cartridge in a point-of-care optical detection system based on a smartphone. We show positive results within the 10-30 minutes range and integrated biological controls on the cartridge. We demonstrate the microfluidic diagnostic with human patient samples, with results that are consistent with the off-cartridge validation. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
We developed a piecewise isothermal nucleic acid test (PINAT) as a platform technology for diagnosing pathogen-associated infections, empowered by an illustrative novel methodology that embeds an exclusive DNA-mediated specific probing reaction with the backbone of an isothermal reverse transcription cum amplification protocol for detecting viral RNA. In a point-of-care format, this test is executable in a unified single-step, single-chamber procedure, leading to seamless sample-to-result integration in an inexpensive, scalable, pre-programmable, and customizable portable device, with mobile-app-integrated interpretation and analytics involving minimal manually operative procedures. The test exhibited a high sensitivity and specificity of detection when assessed using 200 double-blind patient samples for detecting SARS-CoV-2 infection by the Indian Council of Medical Research (ICMR), and subsequently using 170 double-blind patient samples in a point-of-care format outside controlled laboratory settings as performed by unskilled technicians in an organized clinical trial. We also established its efficacy in detecting Influenza A infection by performing the diagnosis at the point of collection with uncompromised detection rigor. The envisaged trade-off between advanced laboratory-based molecular diagnostic procedures and the elegance of common rapid tests renders the method ideal for deployment in resource-limited settings towards catering the needs of the underserved.