Design of Multiplex Polymerase Chain Reaction (PCR) Method for Molecular Detection of Yersinia pestis Bacterium

Objective: Yersinia pestis, the causative agent of the zoonotic plague infection, is a ma- jor public health concern both as a threat and potential bioweapon. The objective of the present study was to establish a uniplex and multiplex - polymerase chain reaction (PCR) test for the specific detection of Y. pestis. Materials and Methods: PCR reactions performed by three pair primers which targeted the caf1 and pla genes located on the pFra and pPst plasmids and the irp2 chromosomal gene located on the ‘pathogenicity island’. After TA cloning of the PCR products, the test’s limit of detection (LOD) was determined. For evaluating the specificity, PCR reactions were performed with negative control bacteria. Results: Assays were performed with the genome of Y. pestis which produced three DNA fragments of the expected sizes 300, 400 and 520 bp which corresponded to the irp2, caf1 and pla genes, respectively. The lower LoD was 370 copy numbers for the caf1 gene and 21 for the pla gene. In PCR reactions that used negative control bacteria, detectable fragments were not observed. Conclusion: Our method clearly discriminated Y. pestis DNA. The rapidity, specificity and sensitivity of this procedure suggest that it can serve as a useful alternative method for the inoculation of laboratory animals or the use of specific culture media for routine plaque surveillance and outbreak investigations. Another vital result of this study was the establishment of Y. pestis molecular detection technique in Iran.