Obtaining Specific Sequence Tags for Yersinia pestis and Visually Detecting Them Using the CRISPR-Cas12a System

Abstract

Three worldwide historical plague pandemics resulted in millions of deaths. Yersinia pestis, the etiologic agent of plague, is also a potential bioterrorist weapon. Simple, rapid, and specific detection of Y. pestis is important to prevent and control plague. However, the high similarity between Y. pestis and its sister species within the same genus makes detection work problematic. Here, the genome sequence from the Y. pestis CO92 strain was electronically separated into millions of fragments. These fragments were analyzed and compared with the genome sequences of 539 Y. pestis strains and 572 strains of 20 species within the Yersinia genus. Altogether, 97 Y. pestis-specific tags containing two or more single nucleotide polymorphism sites were screened out. These 97 tags efficiently distinguished Y. pestis from all other closely related species. We chose four of these tags to design a Cas12a-based detection system. PCR–fluorescence methodology was used to test the specificity of these tags, and the results showed that the fluorescence intensity produced by Y. pestis was significantly higher than that of non-Y. pestis (p < 0.0001). We then employed recombinase polymerase amplification and lateral flow dipsticks to visualize the results. Our newly developed plasmid-independent, species-specific library of tags completely and effectively screened chromosomal sequences. The detection limit of our four-tag Cas12a system reached picogram levels.

Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis

“…In summary, we have developed rapid, ultrasensitive, and specific detection methods for F. tularensis using RT-RPA and RPA-CRISPR/Cas12a assays. Compared with other equally sensitive test assays already reported ( Table 2 ) (Versage et al, 2003 ; Chen et al, 2021 ; Li et al, 2021 ; Lee and Oh, 2022 ), the RPA/CRISPR-based detection of F. tularensis in this study offers a number of advantages, namely, rapid, ultrasensitive, specific, portative, and mild; more importantly, the dually diagnostic methods for F. tularensis can avoid error and missed detection to a greater extent. If it is combined with the nucleic acid-free extraction method, this detection method has a great potential value in clinical rapid diagnosis and biosafety maintenance, especially for on-site diagnosis in the future.…”

CRISPR-Cas Systems-Based Bacterial Detection: A Scoping Review

“…Meanwhile, most of the studies reported the LoD in CFU/mL, of which seven studies showed LoD of 1 CFU/mL. Five studies reported the LoD in molarity [ 12 , 17 , 18 , 28 , 38 , 39 ]. Of these, CAS-EXPAR for the detection of Listeria monocytogenes showed the lowest LoD up to the attomolar level [ 38 ].…”