Molecular identification of agrobacterium tumefaciens containing pCAMBIA 1305.2 plasmid using multiplex PCR and Gold nanoparticles multiplex probe

Document Type: Research Paper

Abstract

Conventional microbiology methods used to detect bacteria include multiple cultures and identification processes, so the results of lab work are painstaking and time-consuming. In recent years, more and more tend to use the diagnostic tests which are based on DNA; hence, DNA diagnostic biosensors have been created to perform DNA identification better. In this study, GUS and hpt genes were used to identify the species of Agrobacterium tumefaciens containing pCAMBIA1305.2 plasmid and to design primers and probes. PCR results for both GUS and hpt genes indicated amplification of two expected fragments. The specificity of the primers designed in Agrobacterium tumefaciens bacteria containing pCAMBIA1305.2 plasmid and negative control samples was evaluated. Synthesis of the gold nanoparticles was performed with a diameter of about 20 nm. Genomic DNA of the bacteria was used to detect the gold nanoparticles and the markers. Finally, using probes designed for GUS and hpt genes, simultaneous detection of the bacteria was done using multiplex probes attached to gold nanoparticles. The results showed a change of color in gold nanoparticles in the presence of the target molecule. In addition, the hybridization probes were examined with target molecules at wavelengths between 400 and 700 nm. The most significant changes occurred in the wavelength ranges of 550 to 650 nm. The results showed that using detectors attached to gold nanoparticles had higher speed and specialty than biochemical and molecular methods, and of course, it had lower costs.

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