James Giese

Rapid Test Kits for Meat and Poultry Product Screening
Unlike traditional microbial methods for detection of foodborne bacteria that use growth in culture media, isolation, and biochemical identification, rapid test kits are designed to make detection and identification of microorganisms faster, more convenient, more sensitive, and more specific.

Using DNA fingerprinting, a USDA microbiologist identifies bacterial isolates in carcass-contamination tracking studies.

Some rapid test kits use biochemical techniques for detection, but many are now antibody- and DNA-based tests. With few exceptions, almost all assays used to detect specific pathogens in foods require some growth in an enrichment medium before analysis. Most of these kits consist of a disposable device containing media or substrates specifically designed to identify a bacterial group or species. With the exception of a few kits where results can be read in 4 hr, most require incubation for 18–24 hr.

According to the Food and Drug Administration, the DNA-based assay formats for detecting foodborne pathogens use probes, the polymerase chain reaction (PCR), and bacteriophages. Probe assays target ribosomal RNA (rRNA), because the higher amount of bacterial rRNA provides an amplified target and gives greater assay sensitivity. The PCR assay utilizes short fragments of DNA or primers that are hybridized to a specific sequence, which is then enzymatically amplified using a thermocycler. PCR can amplify a single copy of DNA by a million times in several hours, reducing the need for cultural enrichment. However, inhibitors in food samples can prevent primer binding and diminish amplification efficiency, so that the extreme sensitivity achievable by PCR with pure cultures is often reduced in food testing. Therefore, some cultural enrichment is still required prior to analysis. The interaction of a phage with its bacterial host has also been used to develop assays for foodborne pathogens.

Another group of rapid tests are based on the binding of antibody to antigen. According to FDA, these constitute the largest group of rapid methods being used in food testing. A variety of formats are used in the antibody assays, including latex agglutination; a modification of latex agglutination known as reverse passive latex agglutination; immunodiffusion; the enzyme-linked immunosorbent assay (ELISA); and immunomagnetic separation (IMS) technology.

ELISA is the most prevalent antibody assay format used for pathogen detection in foods. In this format, 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.

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In IMS, antibodies coupled to magnetic particles or beads are used to capture pathogens from pre-enrichment media. IMS is analogous to selective enrichment, but instead of using antibiotics or harsh reagents that can cause stress injury, an antibody is used to capture the antigen—a much milder alternative. Captured antigens can be plated or further tested using other assays.

Immunoprecipitation or immunochromatography is another antibody-based assay that is based on the technology developed for home pregnancy tests. It is also a “sandwich” procedure, but instead of enzyme conjugates, the detection antibody is coupled to colored latex beads or to colloidal gold. Using only a 0.1-mL aliquot, the enrichment sample is wicked across a series of chambers to obtain results. These assays are extremely simple, require no washing or manipulation, and are completed within 10 min after cultural enrichment.

Besides the DNA- and antibody-based tests, there are a variety of other assays, ranging from specialized media to simple modifications of conventional assays, which result in saving labor, time, and materials. Some, for instance, use disposable cardboards containing dehydrated media, which eliminates the need for agar plates, constituting savings in storage, incubation, and disposal procedures. Others incorporate specialized chromogenic and fluorogenic substrates in media to rapidly detect trait enzymatic activity. There are also tests that measure bacterial adenosine triphosphate (ATP), which (although not identifying specific species), can be used to rapidly enumerate the presence of total bacteria.

Regulatory Concerns
In 2000, the U.S. Dept. of Agriculture’s Food Safety and Inspection Service (FSIS) completed implementation of its rule on Pathogen Reduction/Hazard Analysis and Critical Control Point (HACCP) systems, which was published in the Federal Register of July 25, 1996. Under the regulations, each meat and poultry plant must develop and implement a written plan for meeting its sanitation responsibilities and develop and implement a HACCP plan that systematically addresses all significant hazards associated with its products. In addition, all slaughter plants must regularly test for generic Escherichia coli to verify their procedures for preventing and reducing fecal contamination. Raw products from slaughter plants and plants that grind meat and poultry are subject to Salmonella testing by FSIS. These efforts are directed at reducing microbial contamination over time.

In November 2002, FSIS implemented a directive to reduce Listeria monocytogenes in ready-to-eat meat and poultry products. The directive includes instructions for FSIS sampling and verification steps to ensure that a plant’s HACCP plans and Sanitation Standard Operating Procedures (SSOPs) effectively control the presence of L. monocytogenes in ready-to-eat products. FSIS began testing ready-to-eat products for L. monocytogenes in 1987 because it is the only strain of Listeria that is consistently associated with human illness. At the same time, FSIS also amended segments of its microbiological testing program for E. coli O157:H7. Sections of an FSIS directive exempting some slaughter plants from FSIS’s random E. coli O157:H7 testing will be eliminated. All ground beef plants will become part of the FSIS random verification testing program.

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Applications and Advances
Rapid test kits are designed for use with quality control programs to screen many food samples for the presence of a particular pathogen or toxin. However, according to FDA’s Bacteriological Analytical Manual (www.cfsan.fda.gov/~ebam/bam-toc.html), a positive result by a rapid method is only regarded as presumptive and must be confirmed by standard methods. Confirmation of the positive result may extend the analysis by several days. A screening test indicates if a sample is potentially positive for a pathogen. If a sample tests negative, no further analysis is done. If the screening test indicates a potential positive, further testing is done to confirm the screening results.

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, so 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.

Several companies are offering new products in the meat and poultry testing area.

BioControl, Bellevue, Wash., has introduced the EHEC8™ system, a new enrichment for E. coli O157:H7 that, according to the company, cuts the total testing time for raw beef from 24 hr to less than 9 hr. The new enrichment was developed to help beef processors meet the new FSIS directive regarding E. coli O157:H7 contamination of beef products. The new medium is a significant advancement because it shortens the enrichment time from the 16–24 hr required by other methods to less than 9 hr. This allows product to be released up to two days earlier. 

The system includes a single 8-hr enrichment, using BioControl’s proprietary EHEC8 medium, and either the VIP® for EHEC test or the Assurance® EHEC EIA. In addition to getting product to market faster, the EHEC8 System is an AOAC Official Method for the detection of E. coli O157:H7 in raw and cooked beef. The company says that the EHEC8 system has been extensively tested and validated by independent laboratories during a collaborative study conducted among 30 laboratories representing government as well as private industry in the U.S. and Canada. Results demonstrated that the EHEC8 medium used with either VIP for EHEC or Assurance EHEC EIA was equivalent to the USDA/ BAM reference culture procedure.

The EHEC8 System for raw or cooked beef is a fast, flexible system that increases productivity with its one-step enrichment and short turnaround time. The system is complete, and no expensive antibiotics or additives are necessary, making it very cost-effective.

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Neogen Corp., Lansing, Mich., is offering its Gene-Trak Listeria monocytogenes Assay. The DNA hybridization test employs L. monocytogenes– specific DNA probes and a colorimetric detection system for the detection of this species in food and environmental samples following broth culture enrichment. A sample is considered negative for the presence of L. monocytogenes if the absorbance value obtained is less than or equal to the established cutoff value for the assay. A sample is considered presumptively positive if the absorbance value obtained is greater than the established cutoff value of the assay.

Neogen also offers the Reveal® test for E. coli O157:H7. The test is said to allow the screening of samples for this pathogen following as little as 8 hr of sample enrichment. To perform the test, a 120- mL portion of the enrichment culture is placed into the round sample port of the test device and is wicked through a reagent zone which contains specific antibodies (anti–E. coli O157:H7) conjugated to colloidal gold particles. If antigens are present in the sample, they will bind to the gold-conjugated antibodies. This antigen–antibody complex then leaves the reagent zone and travels through the nitrocellulose membrane which contains a zone of anti-E. coli O157:H7 antibody. The immune complex with gold conjugate is captured and aggregates in this zone, thus displaying a visible line. It is an AOAC official method used by FSIS.

Qualicon Div. of DuPont, Wilmington, Del., offers the BAX® system to screen ready-to-eat products for L. monocytogenes. The screening test uses PCR to amplify DNA. If a pathogen is present in a sample, the system copies the DNA of the pathogen, producing enough DNA to be detected. If amplification occurs, the sample is analyzed further. If no amplification occurs, no further testing is done on the sample. As part of its ongoing process of evaluating procedures to shorten the time it takes to obtain test results while maintaining analytical quality, FSIS adopted this test in April 2002 to screen meat and poultry samples for L. monocytogenes. The agency plans to evaluate the system for Salmonella and E. coli O157:H7 for use in all three of its field service laboratories.

Next Generation
According to FDA, because of the complex formats of rapid tests and the difficulties of testing foods, users must exercise caution when selecting rapid methods and evaluate these tests thoroughly. The technology continues to advance, and the next generation of assays, such as biosensors and DNA chips, that potentially have the capability for near real-time and on-line monitoring of multiple pathogens in foods are already being developed.

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