Obtaining Specific Sequence Tags for <i>Yersinia pestis</i> and Visually Detecting Them Using the CRISPR-Cas12a System
Gang Chen,
Yufei Lyu,
Dongshu Wang,
Li Zhu,
Shiyang Cao,
Chao Pan,
Erling Feng,
Weicai Zhang,
Xiankai Liu,
Yujun Cui,
Hengliang Wang
Affiliations
Gang Chen
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijng 100071, China
Yufei Lyu
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijng 100071, China
Dongshu Wang
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijng 100071, China
Li Zhu
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijng 100071, China
Shiyang Cao
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China
Chao Pan
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijng 100071, China
Erling Feng
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijng 100071, China
Weicai Zhang
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijng 100071, China
Xiankai Liu
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijng 100071, China
Yujun Cui
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China
Hengliang Wang
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijng 100071, China
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.
