Desperately Seeking Salmonella through PCR

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.