Digital PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse

Authors: VELEZ, FRANK - Florida State University; KANDULA, NETHRAJA - Florida State University; BLECH-HERMONI, YOTAM - Qiagen Sciences Inc; Jackson, Charlene; Bosilevac, Joseph - Mick; SINGH, PRASHANT - Florida State University

Submitted to: Food Control
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/8/2024
Publication Date: 7/11/2024
Citation: Velez, F.J., Kandula, N., Blech-Hermoni, Y., Jackson, C.R., Bosilevac, J.M., Singh, P. 2024. Digital PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse. Food Control. https://doi.org/10.1016/j.crfs.2024.100807.
DOI: https://doi.org/10.1016/j.crfs.2024.100807

Interpretive Summary: Strains of Salmonella are a frequent cause of foodborne illness and are known to contaminate poultry products. Most Salmonella testing methods can only detect Salmonella and cannot quantify or estimate the Salmonella load in samples. The aim of this study was to standardize and validate a digital PCR (dPCR) assay to detect and estimate Salmonella levels in chicken rinse samples. Pure culture Salmonella strains were cultured, enumerated, cold-stressed for 48 hours, and used to inoculate whole carcass chicken rinse at 1 – 4 log CFU/30 mL and enriched at 37°C for 5 hours. Undiluted DNA samples with primer and probes targeting the Salmonella-specific invA gene were used for the dPCR assay. The dPCR assay was highly specific, with a limit of detection of 0.001 ng/µL and a limit of quantification of 0.01 ng/µL. The assays detected all cold-stressed Salmonella in inoculated samples following a 5-hour enrichment and accurately estimated the inoculated Salmonella levels. The assay generated reproducible results with minimal sample-to-sample variations, was highly resistant to PCR inhibitors, and showed high DNA tolerance. This dPCR assay standardized in this study is a robust method for the detection and estimation of Salmonella concentration in contaminated food samples. This approach can allow same-day decision-making for poultry processors attempting to maintain limits and controls on Salmonella contamination.

Technical Abstract: Strains of Salmonella are a frequent cause of foodborne illness and are known to contaminate poultry products. Most Salmonella testing methods can only detect Salmonella and cannot quantify or estimate the Salmonella load in samples. Therefore, the aim of this study was to standardize and validate a partitioned-based digital PCR (dPCR) assay for the detection and estimation of Salmonella contamination levels in poultry rinses. Pure culture Salmonella strains were cultured, enumerated, cold-stressed for 48 hours, and used to inoculate whole carcass chicken rinse (WCCR) at 1 – 4 log CFU/30 mL and enriched at 37°C for 5 hours. Undiluted DNA samples with primer and probes targeting the Salmonella-specific invA gene were used for the dPCR assay. The dPCR assay was highly specific, with a limit of detection of 0.001 ng/µL and a limit of quantification of 0.01 ng/µL. The dPCR assay further showed no PCR reaction inhibition up to 5 micrograms of crude DNA extract. The assays accurately detected all cold-stressed Salmonella in inoculated WCCR samples following a 5-hour enrichment. Most importantly, the dPCR copies/µL values, when converted to log, accurately estimated the inoculated Salmonella levels. This dPCR assay standardized in this study is a robust method for the detection and estimation of Salmonella concentration in contaminated food samples. This approach can allow same-day decision-making for poultry processors attempting to maintain limits and controls on Salmonella contamination.