Adhesive Tape-based Sampling and invA-Specific Recombinase Polymerase Amplification for Detection of Salmonella on a Model Surface

Introduction: Previously, we have shown that adhesive tape-based sampling can be combined with fluorescence in situ hybridization (FISH) for rapid detection of Salmonella on produce surfaces. However, the limited sensitivity of FISH does not enable detection of low levels of Salmonella without enrichment. The combination of adhesive tape-based sampling with a method for pathogen-specific nucleic acid amplification could provide a rapid and sensitive solution for screening of food processing surfaces for the presence of Salmonella. 

Purpose: Others have combined tape-based sampling of environmental surfaces with PCR for forensic analysis of short tandem repeats (STRs) from low numbers of human cells. Inspired by this work, we sought to extend our previous method for tape-based sampling for Salmonella to include specific detection of this pathogen using Recombinase Polymerase Amplification (RPA), a rapid isothermal method for nucleic acid amplification. 

Methods: Different levels of Salmonella enterica ser. Typhimurium ATCC 14028 were inoculated onto a model sample surface (a Tryptic Soy Agar plate) and allowed to adhere. FungiTape (Scientific Device Laboratory, Inc.) was used to remove Salmonella cells from this surface, followed by on-tape lysis and DNA extraction using the PrepMan Ultra reagent (Life Technologies). A 10-min isothermal RPA assay (TwistDx, Ltd.) was used to amplify the invA gene, with lateral flow-based detection of the product (Ustar Biotechnologies, Ltd.). Limit of detection was determined using parallel RPA analysis of Salmonella genomic DNA of known concentration.

Results: Our approach enabled detection of Salmonella at levels as low as 10¹ CFU/μl initial inoculum within 30 min. No inhibition of the RPA assay from co-extraction of tape-based chemicals was detected. 

Significance: Our results suggest this approach may enable simple and rapid sampling and molecular detection of Salmonella in support of environmental testing efforts. Further evaluation on surfaces typical of food processing environments (stainless steel, tile, plastic) is warranted.