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.
Highlights
Plague is a fatal infectious disease caused by Yersinia pestis
The Y. pseudotuberculosis 1682 (Yp1682) strain was isolated from a pig in Japan, the Y. pseudotuberculosis 1678 (Yp1678) strain was isolated from a person in Japan, and the Y. pseudotuberculosis 1688 (Yp1688) strain was isolated from a dog in Japan
We downloaded the genomes of 539 Y. pestis strains and 572 other Yersinia species strains from the National Center for Biotechnology Information (NCBI) genome database (Figure 2b)
Summary
Plague is a fatal infectious disease caused by Yersinia pestis. Plague pandemics, of which there have been three from the sixth century AD to the end of the 19th century, have inflicted a heavy toll on human societies. With the increasing number of sequenced strains, the Yersinia bacterial genome database at the National Center for Biotechnology Information (NCBI) has become large and complicated This database contains some non-Y. pestis genome data. Another specific target gene (pla) encodes the pPCP1 plasmidlocated plasminogen activator These unique plasmids, are not present in all Y. pestis strains [12,13]. These 97 tags are sufficient to efficiently distinguish Y. pestis from other closely related species We used these 97 tags to write an automated program to help researchers quickly identify Y. pestis after they have obtained a draft genome sequence. Four tags selected from the aforementioned 97 Y. pestis-specific tags were designed for use with the Cas12a-based detection system in combination with RPA [27,28] and with LFD methodology [29,30] for easy visualization
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