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

Yang You; Pingping Zhang; Gengshan Wu; Yafang Tan; Yong Zhao; Shiyang Cao; Yajun Song; Ruifu Yang; Zongmin Du

doi:10.3389/fmicb.2021.700016

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

Front Microbiol. 2021 Jul 7:12:700016. doi: 10.3389/fmicb.2021.700016. eCollection 2021.

Authors

Yang You¹, Pingping Zhang¹, Gengshan Wu¹, Yafang Tan¹, Yong Zhao¹, Shiyang Cao¹, Yajun Song¹, Ruifu Yang¹, Zongmin Du¹

Affiliation

¹ State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.

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.

Keywords: Cas12a; Yersinia pestis; lateral flow immunochromatographic assay; nucleic acid detection; up-converting phosphor technology.