Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas12a-UPTLFA Platform

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.

Application of CRISPR/Cas Systems in the Nucleic Acid Detection of Infectious Diseases

“…Not only the fluorescence quenching reporter ssDNA probe is used to report the detection result, but some systems use other detection methods, such as Cas12a-UPTLFA, combined with LFA technology and up-converting phosphor nanoparticle (UCP) label-coupled detection methods [ 35 ]. The method used a FAM-biotin probe (UPT will be conjugated to biotin), labeled ssDNA as the reporter, and the results were obtained by scanning using a UPT biosensor.…”

CRISPR-Based Colorimetric Nucleic Acid Tests for Visual Readout of DNA Barcode for Food Authenticity

“…The results showed that the activated Cas12a can more effectively cleave poly A than poly T, thus G-quadruplex with A bases in the space between G-rich sequences can serve as a more effective reporter than that with T as space bases. It is reported that Cas12a preferred to cut the ploy-C reporter, followed by the poly-A and poly-T reporters sequentially . The number of A bases in the space of G-rich sequence also affected the efficiency of Cas12a trans-cleavage, the values of Δ A of 3 and 6 A bases were 0.38 and 0.52, respectively (Figure B).…”

Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms

“…Supported by automated microfluidic mixing, an approach for Ebola virus detection was established using Cas13a and achieved an LoD of 20 pfu/ ml (5.45 × 10 7 copies/ml) of purified Ebola RNA within 5 min (Qin et al, 2019). Cas12a-based biosensing was also developed for the detection of a variety of pathogenic microorganisms, such as Listeria monocytogenes , Cryptosporidium parvum (Yu et al, 2021), Salmonella (Ma et al, 2021), Helicobacter pylori (Qiu et al, 2021), Yersinia pestis (You et al, 2021), E. coli, and Staphylococcus aureus (Bonini et al, 2021). Coupling with a reversible valve-assisted chip, sample preparation, Cas12a reactions, and LAMP was integrated and controlled precisely to perform the detection of V. parahaemolyticus, achieving an LoD of 30 copies/reaction within 50 min (Wu et al, 2021b).…”