Today, one of the most important problems in detection of human pathogens, is lack of positive control. The idea of using hybrid vectors, containing genes of different pathogens, can overcome this limitation. We can design specific primers for each region and use the hybrid vector as positive control sample in PCR. In this research we designed an in silico hybrid vector and relevant primers for detection of Francisella, Variola, Burkholderia and Yersinia.
In this study, fopA, caf1, 16srRNA and HA genes were chosen to be located on the vector, to respectively represent Francisella, Yersinia, Burkholderia and Variolla,. The sequence of these genes were obtained from NCBI in FASTA format and were aligned in BioEdit 18.104.22.168 software for finding conserve region of each gene, then some purposeful changes were applied in the sequence of each gene and the sequences were placed next to each other and the construct was designed. Specific primers were designed for each region using Oligo7, BioEdit, OligoAnalyzer tool as well as NCBI database. Finally, the construct was cloned in PUC57 in SnapGene 22.214.171.124 and PCR was simulated on hybrid vector using designed primers.
Analysis confirmed that conserved regions for each gene were located on hybrid vector, and simulation of PCR proved the accuracy of designed primers.
A genetic simulator hybrid construct with the types of primers required to identify the four factors Francisla, Varivola, Borghuleria and Yersinia has been designed in silico so that such factors could be identified under positive control in emergencies.