The development of species-specific antisense peptide nucleic acid method for the treatment and detection of viable Salmonella

Abstract

Genotypic based detection methods using specific target sites in the pathogen genome can complement phenotypic identification. We report the development of species-specific antisense peptide nucleic acid (PNA) combined with selective and differential enrichment growth conditions for Salmonella treatment and detection. An antisense PNA oligomer targeting the Salmonella ftsZ gene and conjugated with a cell-penetrating peptide ((KFF) 3 K) was exploited to probe bacteria cultured in three different growth media (Muller Hinton broth (MHB), Rappaport-Vassiliadis Soya Peptone Broth (RVS, Oxoid), and in-house modified Rappaport-Vassiliadis Soya Peptone Broths (mRVSs). Also, water and milk artificially contaminated with bacteria were probed. Antisense PNA provided detectable changes in Salmonella growth and morphology in all media and artificially contaminated matrices except RVS. Salmonella was detected as elongated cells. On the contrary, treated Escherichia coli did not elongate, providing evidence of differentiation and selectivity for Salmonella. Similarly, Salmonella probed with mismatched PNAs did not elongate. Antisense oligomers targeted ftsZ mRNA in combination with selective growth conditions can provide a detection strategy for viable Salmonella in a single reaction, and act as a potential tool for bacteria detection in real food and environmental samples.

Development of a fixation-free fluorescence in situ hybridization for the detection of Salmonella species

“…This limitation was likely associated with the presence inhibitors such as milk fats and proteins, which produced unwanted background signals that interfered with the visualization of the hybridized Salmonella cells. In another genotypic method we developed, the treatment and detection of Salmonella in artificially contaminated milk sample using species-specific antisense PNAs proved difficult until a clean-up procedure was incorporated to remove inhibitors, improve PNA cellular bioavailability and detection efficiency [ 37 ]. Unfortunately, we did not consider artificially contaminating Salmonella in fat-free milk to check if the bacterial detection would still be restricted with a lot of background as observed in the undiluted milk sample.…”