Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Rapid Detection of Microbes Using DNA-PNA Hairpin Probes and the DiSC2(5) Dye (#356)

Josh T Ravensdale 1 2 , Ranil Coorey 2 3 , Gary A Dykes 1 2
  1. Department of Health Sciences , Curtin University, Bentley, WA, Australia
  2. Curtin Health Innovation Research Foundation, Curtin University, Bentley, WA, Australia
  3. School of Molecular and Life Sciences, Curtin University, Bentley, WA, Australia

Rapid identification of pathogenic bacteria on meat processing surfaces is of critical importance to the industry. This project aims to investigate the potential of using the hybridisation chain reaction (HCR) to detect bacteria on different surfaces. The 3 main components of the HCR are 2 hairpin loop oligonucleotide probes and sample DNA. In the presence of a target DNA sequence the hairpin probes will bind to the DNA and form large hybrid complexes. We propose using DNA and peptide nucleic acid (PNA) probes in a buffer containing 3,3 diethylthiadicarbocyanine iodide (Disc2(5)) dye to identify target genetic sequences. If the target sequence is present in a sample, hybridisation will occur and the buffer will change from a blue to a purple colour. This procedure does not rely on enzymatic or antibody-antigen reactions and could become a cheap, quick and highly sensitive tool that can be used in the industry. We have synthesised HCR probes designed to bind to the superoxide dismutase gene (sodM) of Staphylococcus aureus and the invasion Protein A gene (invA) of Salmonella Typhimurium. We have found that modifying the sequence length, GC content and including certain sequence patterns in different regions of the probes affects the sensitivity and stringency of the probes when hybridising to positive control “initiator” molecules. As of yet we have not successfully hybridised these probes to the sodM or invA sequence on bacterial DNA. When we increased the length of the initiator sequences to achieve a 10bp overhang on the 5’ and 3’ ends after hybridisation, hybridisation efficacy decreased. Using restriction endonucleases to fragment the genes has produced weak positive signals. We are currently re-optimising and re-designing our probes to get a clearer positive signal. Once the proof of concept has been established these probes can be easily modified for a broad range of genetic screening applications including determining carriage of genetic diseases in patients, and developing antimicrobial resistance gene profiles from clinical samples.