Rapid Detection’s Role in Marine Food

Results

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.

click for large version

Figure 3: Results of inclusivity and exclusivity testing.

Conclusion

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 protected].

References

1. FAO/WHO. 2011. Risk assessment of Vibrio parahaemolyticus in seafood: Interpretative summary and Technical report. Microbiological Risk Assessment Series No. 16. Rome. 193pp
2. 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/