Foodborne pathogen testing is one of the fastest-growing segments of the real-time PCR (qPCR) market. Participants in the market include both qPCR instrument and reagent providers, including Bio-Rad Laboratories, Life Technologies and Roche, as well as companies that specialize in products for food testing, such as BIOTECON, Congen and DuPont Qualicon.

Earlier this year, both bioMérieux and Merck Millipore enlarged their presences in the market with their respective acquisitions of AES Chemunex (see IBO 5/31/11) and Biotest (see IBO 3/31/11). Other companies that have recently expanded their offerings include QIAGEN (see IBO 8/31/10) and Pall (see IBO 07/31/11). Increased attention to microbial contamination of food products as a result of major outbreaks (see IBO 7/15/11) and regulatory actions (see IBO 12/31/10), as well as developments in qPCR technology, are fueling market growth. Market offerings include a wide choice of enrichment methods and PCR chemistries, sold together in kits for the appropriate food matrix. Ease of use, speed and validation are important product differentiators.

PCR technology is the most commonly used molecular technique for foodborne-pathogen testing. The foodborne pathogens for which qPCR assays are most commonly used are Salmonella, Listeria monocytogenes and E. coli 0157. Raw ingredients, pre-processed foods and processed foods are tested using qPCR, as are processing environments. QPCR is typically used for pathogen screening. The advantages of qPCR for pathogen testing in food compared with other molecular testing techniques such as immunoassays include sensitivity, selectivity, detection speed and automation. Among the drawbacks are cost, interference from matrix components and enrichment times.

Developments in qPCR chemistries and extraction and enrichment techniques have addressed concerns about qPCR for foodborne-pathogen testing. Because qPCR detects both live and dead cells in a sample, it can produce false positives. Among the solutions to this problem are the use of reverse-transcriptase PCR (RT-PCR), which measures mRNA instead of DNA, and the development of pretreatment solutions to extract dead cells. To increase the efficiency of pathogen detection, RT-PCR’s sequence-specific detection enables multiplex testing for pathogens. Sample preparation advancements include improvements in bacterial concentration and removal of PCR inhibitors.

DuPont Qualicon offers the BAX System Q7, developed with Applied Biosystems (Life Technologies), which includes a cycler/detection system for testing up to 96 samples, PCR tableted reagents and reagents for lysis extraction. “[I]t has been adopted by the US Department of Agriculture Food Safety and Inspection Service for monitoring beef and poultry,” said Megan DeStefano, global marketing manager for DuPont Qualicon. The System is used throughout the food testing process. “Food manufacturers and processors, along with contract labs and food inspection labs, are typical users of PCR technology as part of their quality control processes.” The System and reagents are also sold by Oxoid, a unit of Thermo Fisher Scientific.

Ms. DeStefano emphasized the BAX System’s ease of use, including the extraction protocol. “Users simply transfer a volume of enriched sample to a tube containing the lysis solution and heat it for a specified time (about 30 minutes for Gram-negative bacteria, longer for Gram-positive bacteria),” explained Ms. DeStefano. The lysate is also used to hydrate the PCR tablets, followed by amplification and detection. “Clear positive or negative results are displayed in about 50 minutes for real-time assays,” she said. “The system does not require advanced skills to operate, and clear yes-or-no results do not require expert interpretation.”

DuPont Qualicon offers several PCR chemistries. Describing the company’s products, Ms. DeStefano told IBO, “Our real-time E. coli O157:H7 and STEC assays use Scorpion probes. Our real-time Campylobacter, Vibrio and Staph aureus assays use Taqman probes.” Last year, the company licensed access to Biosearch Technologies’ BHQ, CAL Fluor and Quasar patents (see IBO 2/28/11). Biosearch also makes Scorpion primer oligonucleotides for DuPont Qualicon. DuPont Qualicon is also addressing evolving demands, such as the “Big Six” (non-0157) Shinga-toxin E. coli pathogens (STEC) (see sidebar). “We expect to release a real-time PCR assay for STEC detection very soon,” she told IBO. DuPont Qualicon has partnered with the USDA to develop a test.

German firm BIOTECON Diagnostics offers the foodproof line of qPCR-based testing kits and extraction products for foodborne pathogens. BIOTECON’s kits have been marketed by Merck KGaA since 2008. The company is distinguished by its close connection to the standardization process, according to BIOTECON’s Christina Harzman, PhD. CEO Kornelia Berghof-Jaeger is the head of the European Committee for Standardization for “PCR For the Detection of Pathogens in Food and Feed.” New products include a gene-specific kit for qualitative detection of E. coli 0104:H4 and a serotype-specific kit for qualitative detection.

BIOTECON’s PCR kits utilize hybridization probes for use with Roche LightCyclers and hydrolysis (5’ nuclease) probes for use with various thermal amplification systems. As Dr. Harzman told IBO, BIOTECON’s kits are open platform, providing for greater flexibility. “[I]f the user has a real-time PCR instrument in which they can select the time, temperature and fluorescent channels, then they are able to use our kits.” For enrichment, the company provides lysis or wash-column extraction kits. “DNA extraction is able to be performed directly to the primary enrichment; no secondary enrichment is required.”

Enrichment and extraction are key steps in qPCR-based food pathogens. Advancements in these areas have broadened the food types with which qPCR can be used most efficiently. “Real-time PCR can be used on all foods, including high-protein foods, high-fat foods, as well as spices and coffee. The key is proper homogenized enrichment and having a DNA-extraction method optimized to the food matrix and organism to be detected, provided the real-time PCR kit is specific for the organism you are looking to detect.” She highlighted BIOTECON’s foodproof Salmonella Detection Kit, stating that “[it] has been validated to work properly on over 750 strains of Salmonella on over 100 different food matrices with certification agencies such as the AOAC, MicroVal and NordVal.”

Although qPCR is much faster than PCR or culture methods, sample preparation time is required. “The limiting factor for pathogen detection by real-time PCR is the required initial enrichment,” explained Dr. Harzman. “As the DNA extraction and real-time PCR can be complete in as little as two hours, the need for rapid results is currently limited by the required sample-enrichment period, which varies by pathogen.” The company’s ShortPrep II Kit provides initial sample enrichment in 20–48 hours and is used with the Listeria monocytogenes Detection Kit (5’ Nuclease), which runs in two hours.

Other problems with qPCR for foodborne pathogen testing have been cross contamination of testing apparatus, leading to false positives, as well as concerns that qPCR cannot detect live cells versus dead cells in sample. Dr. Harzman told IBO that BIOTECON’s tests contain Uracil-N-Glycosylase, which prevents false-positives from cross contamination. She also said, “BIOTECON Diagnostics also offers a product called Reagent D, which prevents false-positives from dead cells.” Reagent D eliminates DNA from dead cells.

Automation has also enabled faster and easier testing workflows. BIOTECON offers the foodproof RoboPrep+ Series for sample preparation and PCR setup. Developed with lab automation firm Xiril, the system is available in six different versions. It is utilized in conjunction with the foodproof Magnetic Preparation Kit 1 for magnetic bead–based extraction. “BIOTECON Diagnostics developed the first robot for automated DNA extraction and PCR setup, which is currently undergoing AOAC and MicroVal validation,” said Dr. Harzman.

Dr. Harzman noted that ISO 16140 I, which addresses qPCR of foodborne pathogens, is currently being revised. Asked about the possible effects of this, she told IBO, “The validation process for reference methods like real-time PCR should become more efficient as well as improvements in their statistical calculations. Also methods for in-house validation of molecular methods are described.” Both changes are likely to help improve utilization of qPCR testing for foodborne pathogens. She also noted, in regard to EU regulations, that more specific CEN/ISO standards are expected to be developed for food-relevant pathogens, such as Clostridium botulinum, Yersinia, Vibrio and food-relevant viruses. “These official methods will also increase acceptance to work with real-time PCR methods, especially in official laboratories, which are very much dependent on such official methods.” As for the US’s Food Safety Modernization Act, she said: “One requirement of the Food Safety Modernization Act is better record keeping and reporting requirements, which through data files generated with real-time PCR cyclers can be quickly and easily incorporated into a company’s record keeping system.”

For foodborne pathogen testing, Bio-Rad offers the iQ-Check q-PCR kits for quantitative analysis and PCR amplification and detection systems. The kits utilize a double-stranded DNA hybridization probe developed by Bio-Rad, buffered peptone water for sample enrichment and a lysis reagent for DNA extraction. Discussing Bio-Rad’s kits, Brad Crutchfield, vice president and group manager of Bio-Rad’s Life Science Group, highlighted the kits’ sensitivity and specificity, international validations and speed. “Due to the increased sensitivity of the kits, users obtain results faster than with competitive systems. For example, Listeria is an organism that grows slowly. Most methods require an enrichment time of 48 hours, whereas the enrichment with the iQ-Check kit is only 24 hours.”

He also noted the user friendliness of IQ-Check kits, which includes software for simplified reaction setup. “We have platforms for low- and high-volume labs. All of our test kits run off of the same protocol so users can run multiple tests at the same time to fit their individual lab requirements.” As a large company, Bio-Rad can also offer a range of products for the complete workflow. “We are one of the only suppliers to the food safety industry that can provide instruments, kits and enrichment media all manufactured by Bio-Rad,” noted Mr. Crutchfield.

A wide variety of foods, both raw and processed, are tested for foodborne pathogen using qPCR. “Examples range from raw foods (beef, poultry, eggs), dairy (milk, cheese), produce (leafy greens, fruit), ready-to-eat products (deli meats, hot dogs), processed foods (prepared meals, peanut butter, bakery products) and pet food,” stated Mr. Crutchfield. In addition to testing of the food, processing facilities are also tested. “In some cases, like the poultry industry, testing is performed on pre-production samples from the bird hatchery environment.”

Asked about the possible impact of the Food Safety Modernization Act, Mr. Crutchfield told IBO, “Now that the FDA has the power to conduct recalls, processors will need to be sure that their food safety plans are up to specification.” He believes that for these applications, PCR offers advantages. “Real-time PCR provides the high level of sensitivity and specificity that is not seen with other technologies.”