Design and construction of an internal control for detection Yersinia pestis using PCR

Yersinia pestis, a Gram-negative, non-motile and Slow growth bacterium belonging to the Enterobacteriaceae family, is the causative agent of plague. It has been classified by CDC in group A of bioterrorism agents due to its high morbidity and mortality and easy person to person dissemination. In spite of sensitivity and High accuracy PCR, false negative results can occur due to the presence of inhibitors in clinical sample. The aim of this study was to construction an internal control for the specific detection of Y.pestis.
In this study, The diagnostic primers for the F1 capsule antigen gene (caf1) and the plasminogen activator gene (pla) were designed using Allele ID 7.6 software. our target region was a conserved fragment in the pla and caf1 genes of Y. pestis. The composit primers for IC were designed using Pichia pastoris AOX1 gene sequences and target gene. The PCR experiment was performed on genomic DNA of Y.pestis. The product was cloned in the pTZ57R/T vector to construct a internal control. Then, the internal control function was evaluated By using diagnostic primers.
Findings: The PCR products of the pla and caf1 genes were 136bp and 117bp respectively on electrophoresis gel and length of IPC were 225bp and 227bp respectively, so there was a significant different between their size.
The results indicate that the IC effective tool for avoid false negative results and confirm the results.