Designing Two Synthetic Constructs for Real Time PCR Detection of Francisella tularensis and Ebola Virus

Mohammadjavad Dehghan Esmat Abadi; Hesam Motalebzadeh; Mahmoud Barati; Mohammadali Yaghobi

doi:10.18502/ajmb.v13i2.5528

Designing Two Synthetic Constructs for Real Time PCR Detection of Francisella tularensis and Ebola Virus

Avicenna J Med Biotechnol. 2021 Apr-Jun;13(2):92-97. doi: 10.18502/ajmb.v13i2.5528.

Authors

Mohammadjavad Dehghan Esmat Abadi¹, Hesam Motalebzadeh², Mahmoud Barati³, Mohammadali Yaghobi¹

Affiliations

¹ Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran.
² Department of Medical Biotechnology, Faculty of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran.
³ Department of Genetics, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran.

Abstract

Background: Generally, timely diagnosis of micro-organisms is very important to prevent many diseases. Many methods can detect micro-organisms like culture-based methods and molecular methods. The molecular methods are usually preferred because they provide fast and reliable results. In some cases, microbial strains are not accessible, and there is no safety to work with them; therefore, synthetic constructs which are designed according to the available sequences in databases can be used as a positive control for detection of them.

Methods: In this study, a synthetic construct was designed for molecular detection of Francisella tularensis (F. tularensis) and the Ebola virus by multiplex real-time PCR reaction. For this, sequences were taken from databases and then multiple alignments were done by software. Also, conventional PCR and two models of real-time PCR (SYBR green and TaqMan) were applied. Finally, multiplex real-time PCR was performed.

Results: The synthetic construct was designed and used for conventional PCR and multiplex PCR. The results of common PCR showed a single band at 148 bp and 167 bp in 1.5% agarose gel stained by ethidium bromide for F. tularensis and Ebola virus, respectively. Also, a dual-band at 148 and 167 bp was observed in multiplex PCR. Results of real-time PCR showed a limit of detection about 0.1 pg of plasmid/μl.

Conclusion: In conclusion, the designed construct can be used as a positive control for an accurate diagnosis of these micro-organisms without any biological danger for laboratory staff. So, this method is useful for diagnosis of these agents in food, water, and blood samples.

Keywords: Ebola virus; Humans; Multiplex polymerase chain reaction; Real-time polymerase chain reaction.