Design and evaluation of nanobiosensor based on Surface Plasmon Resonance (SPR) of gold nanoparticles for rapid detection of V.cholerae genome in water samples

Background and Aim : Vibrio cholerae (cholera) is a curved, motile and gram-negative bacterium that transmits to humans through eating contaminated food and water and is commonly a major health threat to developing and underdeveloped countries worldwide. V.cholerae infection can be confirmed by isolating the organism from the feces on the selected medium, followed by biochemical tests and specific antibodies for serotyping and serogrouping. The aim of this study was to develop a simple, sensitive, and rapid assay for the detection of V.cholerae genome. Methods : ctxA gene was selected as the target then specific probes and primers, targeting this gene were designed. Gold nanorods and fabricated nanoprobes were evaluated and approved by UV-vis (۴۰۰-۹۰۰ nm), zeta potential, FT-IR and TEM electron microscopy instruments. Direct detection of bacteria was performed using PCR, Real-Time PCR and biosensor. Limit of Detection (LOD), sensitivity and specificity of all methods were examined using a recombinant plasmid (pJET۱.۲/ctxA) and finally, the results of all methods were compared with each other.Results : Evaluation of designed primers and probes showed no homology to other bacteria and complete specificity for V.cholerae, then the rod forms of nanoparticles and probe hybridization to GNRs (Nanoprobe production) were confirmed by plasmonic and TEM studies. The specificity of all tests was ۱۰۰%. Based on the recombinant vector used, LOD for Real-Time PCR, Biosensor and PCR were ۱۰ to the power of ۳, ۱۰ to the power of ۳, ۱۰ to the power of ۴ Copy of target gene respectively.Conclusion : Since methods for practical, rapid, simple and sensitive detection of V.cholerae is in great demands, using surface plasmon resonance (SPR) biosensors which are one of the most sensitive optical biosensors could be reserved as an alternative method of detection. Although the sensitivity of Real-Time PCR was higher than that of a biosensor, the biosensor was suitable for screening and diagnosis of V.cholerae due to its specificity, acceptable sensitivity (compared to PCR), and its ability to become a useful tool in clinical laboratories.

کلیدواژه ها:

Vibrio cholerae ، Nanobiosensor ، Surface plasmon resonance ، ctxA