Simultaneous and molecular detection of yersinia and francisella using multiplex-pcr

Nafiseh Pourmahdi,1 Mehdi zeinoddini,2,* Mohamad javad dehghan esmatabadi,3 Fatemeh sheikhi,4

1. Malek Ashtar University of Technology, Tehran, Iran.
2. Malek Ashtar University of Technology, Tehran, Iran.
3. Malek Ashtar University of Technology, Tehran, Iran.
4. Malek Ashtar University of Technology, Tehran, Iran.

Abstract

Introduction

Y. pestis causes plague and f. tularensis lead to tularemia, which are known as newborn and retire diseases. immunological and culture-based detection methods of these bacteria are time-consuming, costly, complicated and biosafety level 3 (bsl3) laboratories are required for working on these bacteria, therefore according to absence of standard strain of these bacteria and difficult genome isolation methods, developing molecular detection methods base on a gene structure as positive control sample is valuable.

Methods

In this research, we designed and construct a plasmid, containing a conserved gene of each bacterium. selected target region for francisella is fopa and for yesinia is caf1(f1 capsule antigen). after that, the construct inserted in puc57 and transformed into e.coli dh5α. after an overnight culture, plasmids extracted and used for multipelex pcr. the pcr products were analyzed in 2% agarose gel.

Results

We expect to see a 107bp band for francisella and a 176 bp band for yersinia. the results showed that amplification from each region was successful and expected bands were observed in electrophoresis.

Conclusion

Some bacteria have complicate immunological and culture-based detection tests, or sometimes we encounter with lack of standard microbial strain for some bacteria, so equipped labs are required for working with these bacteria. molecular detection methods can overcome these limitations. due to these difficulties, we can clone conserved genome regions of these bacteria into other bacteria and use them as positive control samples for molecular detection tests.

Keywords

Francisella, yesinia, multipelex pcr, detection, positive control sample