A Rotatable Paper Device Integrating Reverse Transcription Loop-Mediated Isothermal Amplification and a Food Dye for Colorimetric Detection of Infectious Pathogens.

In this study, we developed a rotatable paper device integrating loop-mediated isothermal amplification (RT-LAMP) and a novel naked-eye readout of the RT-LAMP results using a food additive, carmoisine, for infectious pathogen detection. Hydroxyl radicals created from the reaction between CuSO4 and H2O2 were used to decolor carmoisine, which is originally red. The decolorization of carmoisine can be interrupted in the presence of DNA amplicons produced by the RT-LAMP reaction due to how DNA competitively reacts with the hydroxyl radicals to maintain the red color of the solution. In the absence of the target DNA, carmoisine is decolored, owing to its reaction with hydroxyl radicals; thus, positive and negative samples can be easily differentiated based on the color change of the solution. A rotatable paper device was fabricated to integrate the RT-LAMP reaction with carmoisine-based colorimetric detection. The rotatable paper device was successfully used to detect SARS-CoV-2 and SARS-CoV within 70 min using the naked eye. Enterococcus faecium spiked in milk was detected using the rotatable paper device. The detection limits for the SARS-CoV-2 and SARS-CoV targets were both 103 copies/µL. The rotatable paper device provides a portable and low-cost tool for detecting infectious pathogens in a resource-limited environment.

RT-LAMP assay combining multi-fluorescent probes for SARS-CoV-2 RNA detection and variant differentiation.

Simple and accurate testing tools for SARS-CoV-2 viral RNA detection are essential for the prevention of the spread of the virus and timely governmental actions. Herein, we present a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of ORF1ab and N gene fragments of SARS-CoV-2 in one pot. Using two primer sets and two molecular beacon (MB) probes respectively labelled with different fluorophore, positive results were obtained with a limit of detection of 20 and 2 copies/µL for ORF1ab and N gene fragments, respectively. Moreover, the RT-LAMP based assay was applied to detect single-site differences in S genes using two one-step displacement (OSD) probes targeting wild-type and mutant (P681R mutation was chosen as model) genes. Through that, the wild type strain and P681R mutant variant were well distinguished from each other, and a preliminary observation was also made on other mutations at this site such as P681H. The proposed method has high sensitivity for quantification and high specificity for mutation differentiation. In addition, it does not require accurate sophisticated thermal cycler instrumentation and can be used in clinical settings in resource-limited regions.

Lateral flow-based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use.

The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.

Detection of Coronaviruses Using RNA Toehold Switch Sensors.

A rapid, sensitive and simple point-of-care (POC) nucleic acid diagnostic test is needed to prevent spread of infectious diseases. Paper-based toehold reaction, a recently emerged colorimetric POC nucleic acid diagnostic test, has been widely used for pathogen detection and microbiome profiling. Here, we introduce an amplification method called reverse transcription loop-mediated amplification (RT-LAMP) prior to the toehold reaction and modify it to enable more sensitive and faster colorimetric detection of RNA viruses. We show that incorporating the modified RT-LAMP to the toehold reaction detects as few as 120 copies of coronavirus RNA in 70 min. Cross-reactivity test against other coronaviruses indicates this toehold reaction with the modified RT-LAMP is highly specific to the target RNA. Overall, the paper-based toehold switch sensors with the modified RT-LAMP allow fast, sensitive, specific and colorimetric coronavirus detection.

Recent progress on the diagnosis of 2019 Novel Coronavirus.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Therefore, convenient, timely and accurate detection of SARS-CoV-2 is urgently needed. Here, we review the types, characteristics and shortcomings of various detection methods, as well as perspectives for the SARS-CoV-2 diagnosis. Clinically, nucleic acid-based methods are sensitive but prone to false-positive. The antibody-based method has slightly lower sensitivity but higher accuracy. Therefore, it is suggested to combine the two methods to improve the detection accuracy of COVID-19.