Desperately Seeking Salmonella through PCR

The newest generation of PCR systems used in the detection of Salmonella (AOAC # 050602) benefits from the latest advancements in microbiology and genetic science. It incorporates three levels of specificity to ensure a high level accuracy with the fastest time to results. Highly optimized primers are also used in this system to initiate amplification of DNA specific to Salmonella spp. In addition, a probe-based technology replaces the earlier melt-curve analysis technology, which can lead to ambiguous results. An amplification step copies the Salmonella target DNA, if present, over one billion-fold. The probe binds to the copied DNA product, called “amplicon,” and produces a fluorescent signal by means of a fluorescent label molecule that is detected by the a thermal cycler instrument. With each PCR cycle, the target DNA is copied and the signal increases. The amount of signal emitted is directly related to the quantity of target DNA present in the reaction tube allowing for the ability to quantify positive results in addition to receiving a positive/negative determination of each sample.

In addition to the advanced detection, the technology has broken new ground with a single-step, non-proprietary enrichment media and an enrichment time of less than 24 hours. Salmonella enterica encompasses a large group of gram-negative, enteric bacteria, comprising over 2,300 serotypes 2, making it a challenge for assays to detect all Salmonella, while not detecting common cross reactors. Both antibody assays and earlier PCR systems have lacked the sensitivity and specificity to accomplish this without multiple or proprietary enrichment media.

Additionally, sample prep of food samples is made more challenging by their extremely diverse range of inhibitory biochemicals. A variety of technologies is available to remove inhibitors and concentrate the target including columns, centrifugations, or precipitations, which are not efficient for regular use in food testing. Consequently, most Salmonella assays have included a dilution step to dilute the inhibitors in the sample. While relatively effective in reducing the presence of cross reactors, they result in also diluting target organisms if present thereby compromising sensitivity. The technology utilizes an innovative immuno magnetic separation (IMS) step to capture and concentrate the Salmonella cells from the sample and remove from cross reacting organisms. Utilizing a magnetic 8-channel device adds another level of specificity to the assay. The concentrated target is then processed in a real-time thermo cycler which offers results in 75 minutes. The probe technology allows for the amplification and detection of the target to be observed in “real time” as the amplification is in progress. If the target is present with each heating and cooling cycle the fluorescent curve is plotted on the computer screen. Salmonella positives can be determined when they cross the threshold. Additionally, the real time technology allows for the quantitation of the target. Food processors interested in quantifying the burden of Salmonella in foods can take advantage of the inherent quantitative characteristics. It has been shown that the assay is able to correlate the level of amplified target from the enriched sample with initial contamination levels thus providing an estimated range of contamination for use in trouble shooting or evaluating the effectiveness of pathogen reduction strategies and interventions.

References:

• http://www.ers.usda.gov/briefing/FoodborneDisease/Salmonella.htm
• Calnek-BW, Barnes-MJ, Beard-CW, Mcdougald-LR and Saif-YM (1997). Avian Salmonellosis. In. Diseases of Poultry, 10th ED, 1997.81-129.
• Marckus Jucker, PhD, of BioControl Systems, Inc. (Bellevue, Wash.) can be reached at 425-603-1123.