Purpose: This study was conducted to develop and validate a sample preparation solution for mitigating environmental free nucleic acid detection using the Atlas®Listeria Environmental Detection Assay.
Methods: A phage based processing aid was serially diluted and assayed with the Atlas® Listeria spp. Detection Assay to estimate load of residual analyte in the product. An enzyme treatment to degrade free RNA was investigated and application parameters (concentration, temperature, time, and environment) optimized to evaluate direct and indirect treatment of post-enriched environmental samples by the Atlas® Listeria Environmental Detection Assay in the presence of the processing aid. To validate the optimized solution, 60 environmental swabs were collected from a facility, 30 of which were used to swab 4” x 4” coupons treated with the processing aid and another 30 without treatment for inoculation with Listeria spp. All swabs were enriched and assayed by the Atlas®method, with and without the novel sample preparation, and by cultural analysis.
Results: The processing aid product, alone, contained greater than 1010CFU equivalents of free, residual analyte. Enzyme efficacy at 50U/reaction was markedly improved when samples were prepared with a wash step and optimized reconstitution buffer as compared to direct treatment of enrichment sample. For validation with Atlas®, all 30 uninoculated samples from processing aid treated surfaces were falsely positive by standard assay and 29 were resolved when assayed with the novel sample preparation treatment. From inoculated samples, 22 culture confirmed samples were detected by both the standard and modified assays.
Significance: The described sample preparation solution effectively mitigates free nucleic acid detection by the Atlas® LE Detection Assay while providing an additional tool for troubleshooting environmental positive samples.