Detection of Multiple Foodborne Pathogen Genera in a 96-Well Assay at Ten CFU/g Food within Five Hours

Introduction: While polymerase chain reaction analyzers are replacing culture growth to reduce foodborne pathogen analysis times, they are only effective if there are 10,000 to 100,000 pathogenic cells per gram of sample.  For this reason numerous samples are “pooled” together and added to enrichment broths to achieve such concentrations.  Not only does analysis still take 20 to 30 hours, but if a sample tests positive, all of the individual samples (10 to 25) must now be analyzed.   

Purpose: There is a critical need for a multiplexable analyzer that can rapidly detect foodborne pathogens at 1 CFU per gram of food in multiple samples simultaneously, in a few hours (not days). 

Methods: Twenty-five gram food samples were inoculated with 1 of 3 pathogen genera at 10, 100, or 1,000 CFU/g, then incubated for 4 hours in an enrichment broth.  Filtered samples were then added to antibody functionalized 96-well microplates, which were washed to remove unbound materials.  Silver colloids were then added to the wells to produce surface-enhanced Raman spectra (SERS) using a Raman spectrometer. 

Results: Samples of 10 CFU/g Campylobacter jejuni in chicken, Listeria monocytogenes in cheese, and Salmonella Typhimurium in chocolate were detected within 5 hours using this assay.  Measurements of each pathogen genera in wells functionalized with the antibodies specific to the other two genera required concentrations at least 1,000 times greater to produce a SERS signal.

Significance: The entire time required to detect foodborne pathogens in food or equipment samples at 10 CFU, can be performed in 5 hours, which includes 4 hours of broth enrichment.  As many as 96 samples can be measured simultaneously, allowing 1) the detection of multiple pathogen genera and species, 2) analysis of multiple sample sources, 3) repeat measurements, and 4) reference measurements of control samples.