Simple and Rapid Detection of Burkholderia and Vari olla Using Multiplex-PCR

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 ۱۶srRNA 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 Oligo۷, BioEdit, GeneRunner softwares, Oligo analyzer website and NCBI database. Finally, the construct cloned in PUC۵۷ 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 none-pathogenic. We can use these hybrid vectors as positive control, without any concern.

کلیدواژه ها:

Hybrid construct ، Positive control sample ، PCR ، Burkholderia ، Variolla