Varying amplification efficiency Real-time PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse

Authors: KANDULA, NETHRAJA - Florida State University; VELEZ, FRANK - Florida State University; Jackson, Charlene; Bosilevac, Joseph - Mick; SINGH, PRASHANT - Florida State University

Submitted to: Food Bioscience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/17/2024
Publication Date: 7/26/2024
Citation: Kandula, N., Velez, F.J., Jackson, C.R., Bosilevac, J.M., Singh, P. 2024. Varying amplification efficiency Real-time PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse. Food Bioscience. https://doi.org/10.1016/j.fbio.2024.104777.
DOI: https://doi.org/10.1016/j.fbio.2024.104777

Interpretive Summary: Salmonella enterica is a leading cause of foodborne infection resulting in thousands of hospitalizations each year. Present testing for Salmonella detects the presence or absence of the bacteria and fails to quantify the contamination load in samples. This study aimed to standardize and validate a novel varying amplification efficiency real-time PCR assay for the detection and estimation of broad Salmonella contamination levels. Two Salmonella-specific primer-probe pairs with different amplification efficiencies were tested to detect and differentiate between samples contaminated with low or high levels of Salmonella in the food samples. The standardized multiplex PCR assay was validated with 131 pure culture strains and 260 laboratory-inoculated chicken-rinse samples. The multiplex assay specifically identified all Salmonella strains. The assay detected Salmonella in all inoculated samples following a five-hour enrichment and was able to discriminate the high and low levels in most of the samples. This proof-of-concept developed in this study is a simple approach that will enable the food industry to detect and identify high-risk samples contaminated at higher levels.

Technical Abstract: The most common foodborne infection in the world that leads to hospitalizations is caused by Salmonella enterica. The commonly used Salmonella testing workflow is qualitative and can only detect the presence or absence of Salmonella and fails to quantify the contamination load in samples. This study aimed to standardize and validate a novel varying amplification efficiency real-time PCR assay for the detection and estimation of broad Salmonella contamination levels. Our approach relied on the use of two Salmonella-specific primer-probe pairs (i.e., invA and ttrR) with different amplification efficiencies to detect and differentiate between samples contaminated with low or high levels of Salmonella in the food samples. The standardized multiplex PCR assay was validated with 131 pure culture strains and 260 laboratory-inoculated chicken-rinse samples. The multiplex assay specifically identified all Salmonella strains. The invA and ttrR primer-probe showed amplification efficiency of 128% and 90%, resulting in an analytic sensitivity of 0.01 and 0.1 pg/reaction for pure culture DNA samples, respectively. The assay detected Salmonella in all inoculated samples following a five-hour enrichment and was able to discriminate the high and low levels in most of the samples. This proof-of-concept developed in this study is a simple approach that will enable the food industry to detect and identify high-risk samples contaminated at higher levels.