Y-013. Detection and Screening of Francisella tularensis and its Subspecies Using a Large-Scale, Comparative Genomics Approach

L. Jiang1, A. M. Phillippy2, K. S. McIver2, M. Cai1, S. L. Salzberg2, R. R. Colwell2, I. T. Knight1;
1Canon U.S. Life Sci., INC., Rockville, MD, 2Univ. of Maryland, College Park, MD.

Francisella tularensis, the etiological agent of tularemia in humans and animals, is one of the NIAID Category A Priority Pathogens. Among the four recognized subspecies, F. tularensis subsp. tularensis (Type A) is the most virulent. Accurate methods of distinguishing F. tularensis from other bacteria and among the subspecies are urgently needed. With the availability of hundreds of microbial genome sequences, it is now possible to computationally design PCR-based assays that target genome-specific signature sequences. Here we report the use of this approach to design and validate assays that can be used for rapid and accurate detection of F. tularensis and its subspecies. Genomic signature sequences were discovered using a comparative genomics website, Insignia. A total of 3,757 sequenced genomes, including 13 F. tularensis genome sequences, were compared and more than 50,000 F. tularensis signatures, 20 nucleotides or more in length were found. The longest signatures were used to design 100 different TaqMan assays that differentiate F. tularensis from all other bacterial species and human genomic DNA. Assays were also designed using signatures that were subspecies-specific or specific for the pathogenic subspecies (Types A and B). Real-time TaqMan PCR assays were performed to validate the signatures using genomic DNA from F. tularensis strains, near neighbor strains, non-Francisella isolates and human cells. All the reactions were performed with 40 ng of DNA, in duplicate on an ABI 7500 system. Results showed that 95% of the assays detected all the F. tularensis strains tested with cycle threshold (Ct) values less than 20. The subspecies-specific assays did not display any cross-activity with other subspecies. All the PCR reactions with the non-Francisella isolates and human DNA were negative, with Ct values greater than 50. The results confirmed that a whole genome approach to signature sequence discovery can provide robust targets for PCR-based assays that differentiate F. tularensis from other genomes in the background and that subspecies-specific assays can also be developed using the whole genome comparative approach.