An improved non-crosslinking gold nanoprobe-NASBA based on 16S rRNA for rapid discriminative bio-sensing of major salmonellosis pathogens

• Development of a novel bio-sensing method based on 16S rRNA gold nanoprobe-nucleic acid sequence-based amplification (NASBA).
• Improvement of non-crosslinking gold nanoprobe-NASBA technique.
• Attaining simple, rapid, specific and sensitive nanodetection of major serovars of the Salmonella genus.

In the absence of an appropriate detection method for simple and rapid differentiation of major salmonellosis-causing agents, nano-bio-sensing could provide ideal molecular detection approaches for food-borne pathogens. In addition, the 16S ribosomal RNA-based detection techniques would enhance specificity and sensitivity due to the presence of multiple copies of 16S rRNA per cells and also the presence of hypervariable regions in the genes. In this study, we developed a novel detection technique based on 16S rRNA gold nanoprobe-nucleic acid sequence-based amplification (NASBA) in an improved non-crosslinking mode for nanodiagnosis of the most important serovars of the Salmonella genus: Salmonella enteritidis and Salmonella typhimurium. In that regard, we designed a specific set of primers along with a thiolated oligonucleotide gold nanoprobe for one of the hypervariable regions of 16S rRNA gene. The NASBA-gold nanoprobe method was improved to lower the overall assay time to about 80 min and enhance the specificity and reproducibility of the detection process using various closely related non-Salmonella species as well as some Salmonella serotypes. Finally, the sensitivity of the developed method was determined to be around 5 CFUs Salmonella per amplification tube. The developed nano-bio-sensing method could provide a cost-effective and promising assay especially suited for quick identification of food-borne pathogens in the event of outbreaks.

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