The most common foodborne infection in the world that leads to hospitalizations is caused by Salmonella enterica.Most Salmonella testing workflows are qualitative and only determine the presence or absence of Salmonel...The most common foodborne infection in the world that leads to hospitalizations is caused by Salmonella enterica.Most Salmonella testing workflows are qualitative and only determine the presence or absence of Salmonella,failing to quantify the contamination load present.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.The approach relies 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(1 log CFU/30 mL)or high(2-4 log CFU/30 mL)levels of Salmonella.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 effi-ciency of 128%and 90%,resulting in an analytic sensitivity of 0.01 and 0.1 pg/reaction for pure culture DNA samples,respectively.After a 5-h enrichment,the assay detected Salmonella in all inoculated samples and accurately identified high and low contamination levels in 87%(n=226/260)of samples.This varying amplification efficiency(VAE)assay offers a simple approach enabling the food industry to detect and identify high-risk samples contaminated by Salmonella.展开更多
文摘The most common foodborne infection in the world that leads to hospitalizations is caused by Salmonella enterica.Most Salmonella testing workflows are qualitative and only determine the presence or absence of Salmonella,failing to quantify the contamination load present.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.The approach relies 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(1 log CFU/30 mL)or high(2-4 log CFU/30 mL)levels of Salmonella.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 effi-ciency of 128%and 90%,resulting in an analytic sensitivity of 0.01 and 0.1 pg/reaction for pure culture DNA samples,respectively.After a 5-h enrichment,the assay detected Salmonella in all inoculated samples and accurately identified high and low contamination levels in 87%(n=226/260)of samples.This varying amplification efficiency(VAE)assay offers a simple approach enabling the food industry to detect and identify high-risk samples contaminated by Salmonella.
