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

• Main Authors: Mohammad Soleimani, Fatemeh Eini, Mehri Fallah Raufi, Firuzeh Azari, Shahrokh Farzampour, Ehsan Jamshidian, Alireza Khoshdel, Keivan Majidzadeh
• Format: Article
• Language: English
• Published: Royan Institute (ACECR), Tehran 2010-01-01
• Series: Cell Journal
• Subjects: Yersinia pestis; Diagnosis; PCR
• Online Access: http://www.celljournal.org/library/upload/article/af_43825327Soleimani.pdf

Objective: Yersinia pestis, the causative agent of the zoonotic plague infection, is a majorpublic health concern both as a threat and potential bioweapon. The objective of thepresent 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 targetedthe caf1 and pla genes located on the pFra and pPst plasmids and the irp2 chromosomalgene located on the ‘pathogenicity island’. After TA cloning of the PCR products, the test’slimit of detection (LOD) was determined. For evaluating the specificity, PCR reactionswere performed with negative control bacteria.Results: Assays were performed with the genome of Y. pestis which produced three DNAfragments 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 geneand 21 for the pla gene. In PCR reactions that used negative control bacteria, detectablefragments were not observed.Conclusion: Our method clearly discriminated Y. pestis DNA. The rapidity, specificityand sensitivity of this procedure suggest that it can serve as a useful alternative methodfor the inoculation of laboratory animals or the use of specific culture media for routineplaque surveillance and outbreak investigations. Another vital result of this study was theestablishment of Y. pestis molecular detection technique in Iran.