Rapid and Sensitive Diagnosis of COVID-19 Using an Electricity-Free Self-Testing System.

Rapid and sensitive detection of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for early diagnosis and effective treatment. Nucleic acid testing has been considered the gold standard method for the diagnosis of COVID-19 for its high sensitivity and specificity. However, the polymerase chain reaction (PCR)-based method in the central lab requires expensive equipment and well-trained personnel, which makes it difficult to be used in resource-limited settings. It highlights the need for a sensitive and simple assay that allows potential patients to detect SARS-CoV-2 by themselves. Here, we developed an electricity-free self-testing system based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that allows for rapid and accurate detection of SARS-CoV-2. Our system employs a heating bag as the heat source, and a 3D-printed box filled with phase change material (PCM) that successfully regulates the temperature for the RT-LAMP. The colorimetric method could be completed in 40 min and the results could be read out by the naked eye. A ratiometric measurement for exact readout was also incorporated to improve the detection accuracy of the system. This self-testing system is a promising tool for point-of-care testing (POCT) that enables rapid and sensitive diagnosis of SARS-CoV-2 in the real world and will improve the current COVID-19 screening efforts for control and mitigation of the pandemic.

PddCas: A Polydisperse Droplet Digital CRISPR/Cas-Based Assay for the Rapid and Ultrasensitive Amplification-Free Detection of Viral DNA/RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays have been an emerging diagnostic technology for pathogen diagnosis. In this work, we developed a polydisperse droplet digital CRISPR-Cas-based assay (PddCas) for the rapid and ultrasensitive amplification-free detection of viral DNA/RNA with minimum instruments. LbaCas12a and LbuCas13a were used for the direct detection of viral DNA and RNA, respectively. The reaction mixtures were partitioned with a common vortex mixer to generate picoliter-scale polydisperse droplets in several seconds. The limit of detection (LoD) for the target DNA and RNA is approximately 100 aM and 10 aM, respectively, which is about 3 × 104-105 fold more sensitive than corresponding bulk CRISPR assays. We applied the PddCas to successfully detect severe acute respiratory syndrome coronavirus (SARS-CoV-2) and human papillomavirus type 18 (HPV 18) in clinical samples. For the 23 HPV 18-suspected cervical epithelial cell samples and 32 nasopharyngeal swabs for SARS-CoV-2, 100% sensitivity and 100% specificity were demonstrated. The dual-gene virus detection with PddCas was also established and verified. Therefore, PddCas has potential for point-of-care application and is envisioned to be readily deployed for frequent testing as part of an integrated public health surveillance program.