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Designing Two Synthetic Constructs for Real Time PCR Detection of Francisella tularensis and Ebola Virus
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span> <span style="font-size:10.0pt">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.</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods: </span><span style="font-size:10.0pt">In this study, a synthetic construct was designed for molecular detection of <em>Francisella tularensis</em> (<em>F. tularensis</em>) and the <em>Ebola</em> 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.</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results: </span><span style="font-size:10.0pt">The synthetic construct was designed and used for conventional PCR and multiplex PCR. The results of common PCR showed a single band at 148 <em>bp</em> and 167 <em>bp</em> in 1.5% agarose gel stained by ethidium bromide for <em>F. tularensis</em> and <em>Ebola</em> virus, respectively. Also, a dual-band at 148 and 167 <em>bp</em> was observed in multiplex PCR. Results of real-time PCR showed a limit of detection about 0.1 <em>pg</em> of plasmid/<em>µl</em>.</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion: </span><span style="font-size:10.0pt">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.</span></span></p>
Ebola virus, Humans, Multiplex polymerase chain reaction, Real time polymerase chain reaction
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https://www.ajmb.org/En/Article.aspx?id=40456
https://www.ajmb.org/PDF/En/FullText/40456.pdf
MohammadjavadDehghan Esmat Abadi61731
HesamMotalebzadehDepartment of Medical Biotechnology, Faculty of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran61732
MahmoudBaratiDepartment of Genetics, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran61733
MohammadaliYaghobiFaculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran61734