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Rapid Detection's Role in Marine Food
by Lisa John, Jörg Slaghuis, and Heike Wulff
Traditional microbiological methods for detection of pathogens in food can require up to five days to obtain a simple yes/no result. This time-consuming process slows the workflow, holding the food in quarantine and preventing its release. It can then result in a considerable delay before products can be put into the market. Immunoassays based on the principle of lateral flow technology allow for convenient detection of pathogens within 24 to 48 hours, depending on parameter. These tests are available for a broad range of pathogens and follow a simple “pregnancy test” design to provide results in a quick, readable format and deliver definite results in as little as 20 minutes after sample enrichment.
Lateral flow tests offer all the benefits of traditional testing methods with the addition of simplicity, speed, reliability, and convenience. When used as part of a monitoring program, they allow streamlining of testing protocols, ensuring the safety of finished products and shortening holding times. Lateral flow tests are currently available for:
- Bacillus cereus—Enterotoxins and emetic toxin,
- E. coli O157,
- STEC/EHEC— Verotoxins (Shiga toxins 1 and 2),
- Legionella/Legionella pneumophila,
- Listeria monocytogenes and Listeria Genus, and
This article describes development and evaluation of a lateral flow test for pathogenic Vibrio parahaemolyticus, a major cause of foodborne illness throughout the world, primarily associated with consumption of contaminated raw or undercooked seafood. (Note the lateral flow test for V. parahaemolyticus is not commercially available.)
Approximately 4,500 cases of V. parahaemolyticus infection are reported each year in the U.S. Numbers are expected to increase worldwide due to greater consumption of raw seafood and the globalization of seafood trade.
Thermostable direct hemolysin (TDH) toxin is known as the major virulence factor of V. parahaemolyticus. Standard detection methods of Vibrio parahaemolyticus vary by country, but all are labor intensive and require three to seven days for results. Because raw seafood quickly experiences deterioration, rapid detection methods are necessary for effective identification of possible contamination.
For this application, a Gold Labeled ImmunoSorbent Assay (GLISA), an immunochromatographic rapid test based on lateral flow technology (Figure 1), was used. The lateral flow assay (LFA) detects the toxin TDH using monoclonal gold-labeled antibodies. If the antigen is present, it reacts with the gold-labeled toxin-specific antibodies and migrates to the binding zone. The gold-labeled toxin-specific antibodies then link to a second specific antibody. Due to the gold-labeling, a distinct red line is formed. The rest of the sample continues to migrate to the control zone and links to a third antibody-specific antibody. The red line formed in the control zone demonstrates that the test is functioning correctly.
Three studies were performed to evaluate the assay for TDH toxin:
- Limit of detection,
- Inclusivity/exclusivity, and
- Evaluation with artificially contaminated food samples.
To establish the limit of detection, four different TDH positive strains of V. parahaemolyticus pure cultures were diluted and tested with the LFA. Pure TDH was also tested.
Inclusivity and exclusivity of the LFA were evaluated by testing a total of 102 isolates and reference strains. Bacteria were cultured in Peptone water (acc. to ISO 6579) plus 2 percent sodium chloride pH 8.5 or in CASO Broth for 18 to 24 hours at 37 degrees Celcius. A total of 160 microliters (μL) of suspension was transferred onto the sample port of the test device. The result was read after 30 minutes.
Because raw seafood quickly experiences deterioration, rapid detection methods are necessary for effective identification of possible contamination.
For evaluation with artificially contaminated food samples, fish and seafood products (oysters, shrimp, and sushi, n=90 total) were spiked with a TDH-positive V. parahaemolyticus strain and analyzed comparatively by the developed test and the reference method according to ISO/TS 21872-1:2007 (Figure 2). For inoculation, stressed and non-stressed cultures were used. Samples were enriched directly following inoculation or after storage for seven days at -20 degrees Celcius. Enrichments were incubated for eight hours and 24 hours. Centrifugation of the sample was evaluated as a pre-sample treatment for performance improvement; 160 μL of sample was transferred to the LFA. The result was read after 30 minutes.
The detection limit of TDH was 125 picograms (pg)/mililiter (ml) and 3.3 x 106 to 1.9 x 107 colony forming unit (cfu)/ml for TDH-positive V. parahaemolyticus, strain-dependent. The LFA achieves an inclusivity rate of 81 percent and exclusivity rate of 100 percent. For the inclusivity six tdh-gene positive V. parahaemolyticus isolates were tested negative by LFA (Figure 3). They are of environmental origin (e.g. seawater and zooplankton) and were tested for TDH production by Latex agglutination test KAP-RPLA (Denka Seiken, Japan). Two of them showed no agglutination (TDH negative) and therefore were excluded in the LFA inclusivity rate calculation.
For fresh food, detection rate of the LFA after 24 hour incubation, in combination with no centrifugation step, was significantly lower than the rate obtained by other methods. In the group of frozen samples, detection after 24 hour enrichment (independent from centrifugation step) was significantly higher than after eight hour enrichment.
For both fresh and frozen food types, 100 percent sensitivity was achieved by LFA after 24 hour enrichment in combination with sample centrifugation. Performance was equivalent to the ISO/TS 21872-1:2007 reference method (100 percent sensitivity) and time-to-result was achieved four days faster. The preliminary centrifugation treatment of the sample significantly increases the detection rate (p=0.035). None of the negative controls were contaminated with TDH-positive V. parahaemolyticus, but sporadically with TDH-negative V. parahaemolyticus. All negative controls reacted negatively by LFA. Therefore, the specificity of the LFA was 100 percent.
Food experiments with artificially contaminated seafood samples showed that TDH-positive V. parahaemolyticus was reliably detected in inoculation concentrations of 101 to 102 cfu/gram in fresh food and 103 to 104 cfu/gram in frozen food after 24 hour incubation.
A GLISA for the detection of pathogenic V. parahaemolyticus in food was developed by targeting the toxin TDH. The detection limit of TDH was 125 pg/ml and 3.3 x 106 to 1.9 x 107 cfu/ml for TDH-positive V. parahaemolyticus. In internal studies (n=102), a sensitivity of 81 percent and specificity of 100 percent was determined for the developed test.
Experiments show that V. parahaemolyticus in seafood can be detected much faster using lateral flow technology than with traditional methods. Detection was completed in 24 hours with enrichment plus one hour sample pretreatment and assay performance compared to three to seven days for standard detection methods.
John, Slaghuis, and Wulff all work in the area of biomonitoring research and development at Merck Millipore in Darmstadt, Germany. John is manager of immunological microbiology group; Slaghuis is director, head of research and development biomonitoring; and Dr. Wulff is a research scientist in immunological microbiology. Reach them at email@example.com.
- FAO/WHO. 2011. Risk assessment of Vibrio parahaemolyticus in seafood: Interpretative summary and Technical report. Microbiological Risk Assessment Series No. 16. Rome. 193pp
- Centers for Disease Control and Prevention (2012). CDC Estimates of Foodborne Illness in the United States. Retrieved July 29, 2013, from www.cdc.gov/nczved/divisions/dfbmd/diseases/vibriop/