Abstract
The recent discovery of collateral cleavage activity of class-II clustered regularly interspaced short palindromic repeats–CRISPR-associated protein (CRISPR-Cas) makes CRISPR-based diagnosis a potential high-accuracy nucleic acid detection method. Colloidal gold-based lateral flow immunochromatographic assay (LFA), which has been combined with CRISPR/Cas-based nucleic detection, usually associates with drawbacks of relative high background and the subjectivity in naked-eye read-out of the results. Here, we developed a novel system composed of Cas12a-based nucleic acid detection and up-converting phosphor technology (UPT)-based LFA (UPT–LFA), termed Cas12a-UPTLFA. We further demonstrated the utility of this platform in highly sensitive and specific detection of Yersinia pestis, the causative agent of the deadly plague. Due to high infectivity and mortality, as well as the potential to be misused as bioterrorism agent, a culture-free, ultrasensitive, specific, and rapid detection method for Y. pestis has long been desired. By incorporating isothermal recombinase polymerase amplification, the Cas12a-UPTLFA we established can successfully detect genomic DNA of Y. pestis as low as 3 attomolar (aM) and exhibited high sensitivity (93.75%) and specificity (90.63%) for detection of spiked blood samples with a detection limit of 102 colony-forming unit per 100 μl of mouse blood. With a portable biosensor, Cas12a-UPTLFA assay can be operated easily by non-professional personnel. Taken together, we have developed a novel Cas12a-UPTLFA platform for rapid detection of Y. pestis with high sensitivity and specificity, which is portable, not expensive, and easy to operate as a point-of-care method. This detection system can easily be extended to detect other pathogens and holds great promise for on-site detection of emerging infectious pathogens.
Highlights
Several promising clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems called Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), one-HOur Low-cost Multipurpose highly Efficient System (HOLMES), DNA endonuclease-targeted CRISPR trans reporter (DETECTR), and Cas14DETECTR (Gootenberg et al, 2017; Chen et al, 2018; Harrington et al, 2018; Li et al, 2018) wereDetection of Yersinia pestis by Cas12a-UPTLFA established
When using Y. pestis genome DNA (3 × 104 fM) as the samples to be detected, about 300 relative fluorescence unit (RFU) signal intensity could be yielded, and no positive signal was found for the samples at lower concentrations (Supplementary Figure 2)
The same batch of spiked blood samples was subjected to quantitative PCR (qPCR) analysis of pla gene, and we found that the lowest concentration that can be detected was 100 CFU per sample, the same with the detection limits of Cas12a-UPTLFA (Supplementary Figure 7)
Summary
Several promising clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems called Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), one-HOur Low-cost Multipurpose highly Efficient System (HOLMES), DNA endonuclease-targeted CRISPR trans reporter (DETECTR), and Cas14DETECTR (Gootenberg et al, 2017; Chen et al, 2018; Harrington et al, 2018; Li et al, 2018) wereDetection of Yersinia pestis by Cas12a-UPTLFA established. Activated Cas12a nucleases indiscriminately cleave the single-stranded DNA (ssDNA) reporter that is labeled with the fluorophore and fluorophore quencher at the both sides, leading to emitting a detectable fluorescent signal (Chen et al, 2018). These methods have the potential to fit all the affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end-users (ASSURED) criteria for nucleic acid detection proposed by the World Health Organization (Peeling et al, 2006). CRISPR/Cas-based detections coupled with colloidal gold strip have been developed to be point-of-care testing (POCT) tools and employed to detect many pathogens, such as Ebola, Lassa, Zika, dengue, human papillomavirus (HPV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Chen et al, 2018; Gootenberg et al, 2018; Barnes et al, 2020; Patchsung et al, 2020)
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