Design and construction of a positive control for molecular diagnosis of Coxiella burnetii
Keywords:
Positive control constructs, Coxiella burnetii, molecular detection, nested-PCRAbstract
Background: Bacterial infections are one of the most important issues in public health and could lead to death in many countries. Molecular methods for detection of these serious infections are fast and reliable, but there is a problem for the diagnosis of some cases of infectious agents, like high-virulence bacteria or rarely founds bacteria. Therefore, designing plasmid DNA construct as positive control is a smart way to solve the diagnosis problem in these cases.
Objectives: The aim of this work was to design and generate a construct to be used as positive control for amplification in the molecular detection assay of Coxiella burnetii (C. burnetti).
Methods: A plasmid construct for C.burnetii and 2 sets of primers were designed. The designed construct contains a specific region of the com1 gene for the diagnosis of C.burnetii. Finally, the designed construct was used as a template in a nested – polymerase chain reaction (PCR) reaction to validate its usefulness.
Results: PCR reaction was performed and 2 specific bands at 580 and 300 bp were observed in 2% agarose gel electrophoresis stained by KBC power load. Also, the sensitivity of the reaction was determined to be about 2.1 x 10-5 ng/L of DNA.
Conclusion: The designed construct could be used as a positive control for a fast and reliable diagnosis of C.burentii. Further, this method can similarly be used for other microorganisms.
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