Simple and Rapid Detection of Yersinia Pestis and Francisella Tularensis using Multiplex-PCR

Yersinia pestis and Francisella tularensis cause plague and tularemia, which are known as diseases of the newborn and elderly, respectively. Immunological and culture-based detection methods of these bacteria are time-consuming, costly, complicated and require advanced equipment. We aimed to design and synthesize a gene structure as positive control for molecular detection of these bacteria.
Conserved regions of each bacterium were determined. A fragment containing the fopA and caf1 genes (conserved genes of F. tularensis and Y. pestis, respectively) was artificially synthesized, cloned into the pUC57 vector (pUC-fopA-caf1), transformed into E. coli DH5α, and used in a multiplex PCR assay. The sensitivity of this assay was examined by serial dilution of the extracted plasmid, whereas the specificity was examined using genomes of Escherichia coli, Salmonella typhi, Enterobacter aerogenes, Vibrio cholerae as templates. Finally, PCR products were analyzed in agarose gel electrophoresis.
As expected, our analysis showed a clear dual band in the size range of 107 bp to 176 bp, confirming the presence of fopA and caf1 genes. Another 351 bp band was detected due to amplification being dependent on the forward primer of fopA and the reverse primer of caf1. Optimization of the PCR protocol reduced the amplification of this 351 bp band. The sensitivity of this assay was determined to be 36×10 -3 ng/µl and the selectivity test confirmed the specificity of this method is appropriate for the detection of target genes.
This multiplex PCR method could be used in research laboratories for identification of these important pathogens.