Journal
ANALYTICAL CHEMISTRY
Volume -, Issue -, Pages -Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.2c03590
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Funding
- National Key Research and Development Program of China [2021YFF0703300]
- National Natural Science Foundation of China [31770849]
- Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong Province [2020B1212060077]
- Zhejiang Provincial Department of Agriculture and Rural Affairs [2022SNJF078]
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In this study, a polydisperse droplet digital CRISPR-Cas-based assay (PddCas) was developed for rapid and ultrasensitive detection of viral DNA/RNA. The method demonstrated high sensitivity, with a limit of detection of 100 aM for target DNA and 10 aM for target RNA. It also showed 100% sensitivity and specificity in detecting SARS-CoV-2 and HPV 18 in clinical samples.
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 x 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.
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