Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples

Abstract

The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.

Dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for colorimetric and point-of-care determination of real SARS-CoV-2

“…We prepared 20 positive samples and 20 negative samples from a patient. Many false-negative cases have been reported when using RT-LAMP to diagnose COVID-19 patients [ 35 – 37 ]. In contrast, our dLig-LAMP system provided 100% true-negatives through both colorimetric detection and absorbance spectral measurements.…”

RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses

“…These conditions can lead to death in severe cases (Mahony, 2008;Rebelo-de-Andrade et al, 2010;Tregoning and Schwarze, 2010;Mahony et al, 2011;To et al, 2017). Various methods have been applied to detect these viruses, including isothermal nucleic acid amplification and PCR-based amplification technique (Zhao et al, 2015;Lim et al, 2018;Pumford et al, 2020;El-Tholoth et al, 2021;Ganguli et al, 2021;Zhang et al, 2021). RAA can accomplish sample detection in 30 min with a sensitivity of a single copy; however, the RAA probe is too long (46-52bp) and, therefore, difficult to design.…”

Microfluidic point-of-care device for detection of early strains and B.1.1.7 variant of SARS-CoV-2 virus

“…In the protocol, the lysed sample is mixed with TE buffer (5% Tween 20), as this buffer has demonstrated to improve sensitivity of the assay 22 while simultaneously diluting the sample by reducing saliva viscosity. 31 Once the mixture enters the amplification-detection reservoirs, both the inlet and outlet of the microfluidic cartridge are sealed using putty and caps. The temperature of the RT-LAMP amplification can result in movement of the solution in the cartridge.…”