Background: 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. Materials and Method: 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. Results: 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. Conclusion: This multiplex PCR method could be used in research laboratories for identification of these important pathogens.
Background: Todays, 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 a hybrid vector and relevant primers for detection of Variolla and Burkholderia. Materials and Methods: In this study 16srRNA and HA genes were chosen to be located on the vector, to represent of Burkholderia and Variolla, in respectively. The sequence of these genes obtained from NCBI in FASTA format and aligned in BioEdit 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 designed for each region using Oligo7, BioEdit, GeneRunner softwares, Oligo analyzer website and NCBI database. Finally, the construct cloned in PUC57 in SnapGene and PCRsimulated on hybrid vector using designed primers. Results: Analysis confirmed that conserved region for each gene is located on hybrid vector for each pathogen, and simulation of PCR proved the accuracy of designed primers. Conclusion: Hybrid vectors design contain similar sequence of pathogens genome but they are nonepathogenic. We can use these hybrid vectors as positive control, without any concern.
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