Design and Production of a Chimeric Construct as a Genetically Positive Control for the Detection of Burkholderia and Yellow Fever Virus Using the Real-Time PCR

Abstract

Background: Burkholderia bacteria are species from protobacter and the pathogens of this species include Burkholderia mallei and pseudomallei causing glanders. The yellow fever virus is a blavovirus that has a single-stranded polyribonucleotide genome (positive strand). Yellow fever is a type of acute viral disease, also called black vomit. Even though considerable advances have been made in the development and use of molecular techniques for the detection of pathogens such as Burkholderia species and yellow fever, a major problem in these techniques is lack of proper positive control. Recently developed genetically modified vectors and simulator construct have provided engineered tools that can be used as a positive control in molecular diagnosis. Methods: In the current study, we first designed and produced chimeric simulator constructs, containing specific genes from Burkholderia mallei, pseudomallei, and yellow fever virus. Then, the operational efficiency of the designed structures was evaluated using a single and dual-mode quantitative (using SYBER Green) and qualitative (using the TaqMan probe) Real-time PCR. Results: The minimum and maximum sensitivity of designed construct for Burkholderia and yellow fever detection was indicated to be 0.1 pg and 10 ng, respectively. Conclusions: Our findings suggest that this positive control constructs can be used for development of new diagnostic kits.