• In Silico design of a hybrid structure as positive control for simultaneous detection of Francisella tularensis and Variolla by PCR method
  • Mona Simkhah,1 Mohammad Javad Dehghan Esmatabadi,2,* Mehdi Zeinoddini,3 Nafiseh Pourmahdi,4
    1. Malek Ashtar University of Technology
    2. Malek Ashtar University of Technology
    3. Malek Ashtar University of Technology
    4. Malek Ashtar University of Technology


  • Introduction: One of the most important problems in detecting pathogens (bacteria, viruses and…) is lack of positive control samples. To eliminate this challenge, designing structures involving conserve region of different pathogens genome, is a functional solution. By assembling these structures on a vector and designing specific primers for each agent, we will have hybrid vectors which can be used as positive control samples for simultaneous and multiple detection by PCR. Francisella tularensis and Variolla are known as a threat for public health and can cause high rate of mortality. Rapid diagnosis of these pathogens, lead to reduce the loss.
  • Methods: In this research, we designed a hybrid structure as positive control for simultaneous detection of Francisella tularensis and Variolla in PCR. fop A gene of Francisella tularensis and HA gene of Variolla were chosen to be present in our vector. Sequence of each gene was obtained from NCBI and aligned in BioEdit version 7.0.5.3 to access the conserve region of each gene. A 100bp region containing EcoRI and NotI restriction sites, and a 48bp region containing BamHI and XhoI restriction sites, were designed to be placed respectively inside and between the genes. According to this, size of fopA gene in actual is 110bp and in construct is 210bp and and HA gene size in actual is 233bp but in construct is 333bp. Specific primers for each region were designed in Oligo versin7. Specifity of primers were confirmed by NCBI Primer Blast. For multiplex PCR detection of these agents, primers were analyzed in Oligoanalyzer. We examined this construct and primers by PCR simulation of SnapGene version 3.2.1.0.
  • Results: we designed a hybrid construct and specific primer for simultaneous detection of Francisella tularensis and Variolla, which can be used as positive control sample in PCR. we examined our construct and primers in SnapGene PCR simulation and observed desired PCR bands.
  • Conclusion: we can design hybrid construct for different human pathogens and use them as positive control samples in PCR detection methods. This leads to rapid, accurate and simultaneous detection with no need of any complicated equipment and laboratories.
  • Keywords: Francisella tularensis, Variolla, Hybrid Vector, Positive Control Sample, PCR