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From: Food Quality & Safety magazine, June/July 2006

Desperately Seeking Salmonella

Detection Methods Utilizing PCR Offer a Higher Degree of Specificity and Ease of Use

by Marckus Jucker, PhD

Salmonella is a group of foodborne bacterial pathogens causing acute gastrointestinal illness around the world. FDA estimates that 2 to 4 million people are sickened by Salmonellosis in the U.S. annually, resulting in approximately 600 deaths in the U.S. and 200 in the EU, with the highest fatality rates in young children and the elderly1.

The CDC estimates that 95 percent of Salmonella infections are acquired through contaminated foods, including raw meats, poultry and produce. Medical costs and lost productivity are the greatest component of Salmonella infections (approximately $3 billion annually; ref 1), recently leading to greater regulatory concern. In February, Dr. Richard Raymond, the USDA’s under secretary for food safety, announced an initiative by the agency’s Food Safety and Inspection Service (FSIS) to reduce the presence of Salmonella in raw meat and poultry products, with a focus on establishments with higher levels of Salmonella.

With this increased scrutiny on Salmonella, manufacturers of rapid detection methods have been challenged with developing new, innovative methods that better address the industries needs of greater accuracy and faster results in Salmonella testing. One method has been developed specifically to meet these requirements.

The first rapid detection methods for Salmonella in foods were antibody-based assays. These assays are still widely used today but have drawbacks in terms of time to results and specificity. Antibody based Salmonella assays require pre-enriching the test sample in a non-selective media, followed by a transfer to a selective media. This procedure can be labor intensive and requires at least 2 days. Antibody based methods in general are effective in detecting Salmonella, but can also detect non-Salmonella cross-reactors with specifically Citrobacters, which can be an irritant to food producers. Detection methods that utilize genetic amplification (PCR) offer a higher degree of specificity and in some cases their ease of use and speed can be extremely advantageous to food manufacturers. PCR is a powerful technology that relies on amplification of the target for detection. Amplification is an exponential process whereby one copy of target DNA is replicated to two, then four, etc., until, after a short time, millions of copies of a unique DNA sequence have been created. As a result, PCR has the ability to quickly detect the presence of just a few target organisms. The process of identifying and copying DNA segments is occurs through a series of controlled temperature changes, known as thermal cycling.

In the PCR process, the DNA target sequence is first identified by a primer — a single strand of DNA with a specific sequence complementary to a portion of the target DNA which is then amplified. Early versions of PCR relied on primers and electrophoresis gels for specificity. Interpreting results required opening the amplification tubes after PCR to pipette amplicon into electrophoresis gels, and subjective reading of the gels. This open tube format was prone to contamination and was never broadly adopted by the food industry.

The second generation of PCR systems, introduced in the late 1990s, provided some improvements. Now the entire PCR reaction, including detection, occurred inside of a PCR tube, eliminating the need for electrophoresis gels. In place of gels, detection was performed by use of a non-specific DNA binding fluorescent dye, such as SYBR Green. SYBR Green is considered non-specific as it will bind to any double stranded DNA, not only the target. This dye disassociates from DNA it is bound to at various temperatures, producing a melt curve which must be analyzed to distinguish the presence of the target from other amplified DNA. Detection of Salmonella in particular was often difficult due to the complexity of the melt curve product by Salmonella. Additionally, this generation of PCR systems did not offer significant advantages in speed.

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:

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