Rapid Detection’s Role in Marine Food

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,
• Campylobacter,
• E. coli O157,
• STEC/EHEC— Verotoxins (Shiga toxins 1 and 2),
• Legionella/Legionella pneumophila,
• Listeria monocytogenes and Listeria Genus, and
• Salmonella.

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.)

Background

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.

click for large version

Figure 1: Principle of Lateral Flow Tests.

Methods

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.

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.