en زیست شناسی مولکولی Molecular biology پژوهشي Research <div style="text-align: justify;"><strong>Background: </strong><em>Yersinia pestis</em> and <em>Francisella tularensis </em>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.<br> <strong>Materials and methods:</strong> Conserved regions of each bacterium were determined. A fragment containing the <em>fopA</em> and <em>caf1</em> genes (conserved genes of <em>F. tularensis</em> and <em>Y</em>. <em>pestis</em>, respectively) was artificially synthesized, cloned into the pUC57 vector (pUC-fopA-caf1), transformed into <em>E. coli</em> DH5&alpha;, 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 <em>Escherichia</em><em> coli</em><em>, Salmonella typhi,</em>&nbsp;<em>Enterobacter aerogenes</em>,<em> Vibrio cholerae </em>as templates. Finally, PCR products were analyzed in agarose gel electrophoresis.<br> <strong>Results: </strong>As expected, our analysis showed a clear dual band in the size range of 107 bp to 176 bp, confirming the presence of <em>fopA</em> and <em>caf1</em> genes. Another 351 bp band was detected due to amplification being dependent on the forward primer of <em>fopA</em> and the reverse primer of <em>caf1</em>. Optimization of the PCR protocol reduced the amplification of this 351 bp band. The sensitivity of this assay was determined to be 36&times;10 ^-3ng/&micro;l and the selectivity test confirmed the specificity of this method is appropriate for the detection of target genes.<br> <strong>Conclusion: </strong>This multiplex PCR method could be used in research laboratories for identification of these important pathogens.</div> Francisella, Yesinia, Multipelex PCR, Detection, Positive Control Sample 28 37 http://rmm.mazums.ac.ir/browse.php?a_code=A-10-903-3&slc_lang=en&sid=1 Nafiseh Pourmahdi npourmahdi@gmail.com 10031947532846007712 10031947532846007712 No Malek Ashtar University of Technology Mehdi Zeinoddini zeinoddini@modares.ac.ir 10031947532846007713 10031947532846007713 Yes Malek Ashtar University of Technology Mohamad Javad Dehghan Esmatabadi mdehghan@mut.ac.ir 10031947532846007714 10031947532846007714 No Malek Ashtar University of Technology Fatemeh Sheikhi sheikhiii12345@gmail.com 10031947532846007715 10031947532846007715 No Malek Ashtar University of Technology