Rapid Methods

The rapid detection of pathogens and other microbial contaminants in food is critical for ensuring the safety of consumers. Traditional methods to detect foodborne bacteria often rely on time-consuming growth in culture media, followed by isolation, biochemical identification, and sometimes serology. Recent advances in technology make detection and identification faster, more convenient, more sensitive, and more specific than conventional assays -- at least in theory. These new methods are often referred to as "rapid methods", a subjective term used loosely to describe a vast array of tests that includes miniaturized biochemical kits, antibody- and DNA-based tests, and assays that are modifications of conventional tests to speed up analysis. Some of these assays have also been automated to reduce hands-on manipulations. With few exceptions, almost all assays used to detect specific pathogens in foods require some growth in an enrichment medium before analysis.

There are many DNA-based assay formats, but only probes, PCR and bacteriophage have been developed commercially for detecting foodborne pathogens. Probe assays generally target ribosomal RNA (rRNA), taking advantage of the fact that the higher copy number of bacterial rRNA provides a naturally amplified target and affords greater assay sensitivity.

The basic principle of DNA hybridization is also being utilized in other technologies, such as the polymerase chain reaction (PCR) assay, where short fragments of DNA (probes) or primers are hybridized to a specific sequence or template, which is then enzymatically amplified by Taq polymerase using a thermocycler. Theoretically, PCR can amplify a single copy of DNA by a million fold in less than 2 hrs; hence its potential to eliminate, or greatly reduce the need for cultural enrichment. However, the presence of inhibitors in foods and in many culture media can prevent primer binding and diminish amplification efficiency, so that the extreme sensitivity achievable by PCR with pure cultures is often reduced when testing foods. Therefore, some cultural enrichment is still required prior to analysis.

The enzyme-linked immunosorbent assay (ELISA) is the most prevalent antibody assay format used for pathogen detection in foods. Usually designed as a "sandwich" assay, an antibody bound to a solid matrix is used to capture the antigen from enrichment cultures and a second antibody conjugated to an enzyme is used for detection. The walls of wells in microtiter plates are the most commonly used solid support; but ELISAs have also been designed using dipsticks, paddles, membranes, pipet tips or other solid matrices.

Applications and Limitations of Rapid Methods
Almost all rapid methods are designed to detect a single target, which makes them ideal for use in quality control programs to quickly screen large numbers of food samples for the presence of a particular pathogen or toxin. A positive result by a rapid method however, is only regarded as presumptive and must be confirmed by standard methods. Although confirmation may extend analysis by several days, this may not be an imposing limitation, as negative results are most often encountered in food analysis. Most rapid methods can be done in a few minutes to a few hours, so they are more rapid than traditional methods. But, in food analysis, rapid methods still lack sufficient sensitivity and specificity for direct testing; hence, foods still need to be culture-enriched before analysis. Although enrichment is a limitation in terms of assay speed, it provides essential benefits, such as diluting the effects of inhibitors, allowing the differentiation of viable from non-viable cells and allowing for repair of cell stress or injury that may have resulted during food processing.