Comparison of real-time PCR and ELISA for the detection of crustacean shellfish allergens.

Food allergies are a significant public health concern, and crustacean shellfish represent one of the major FDA regulated food allergens. Allergic individuals must avoid foods containing crustaceans, and this necessitates highly sensitive and accurate detection methods. Two of the major methods used are protein-based ELISA and DNA-based real-time PCR. In order to properly compare these very different methodologies, we used identical split samples for a side-by-side comparison and analysed them using four different real-time PCR methods and two different commercial ELISA kits. Three real-time PCR assays targeting the mitochondrial 12S genes of shrimp, crab, and lobster were compared to a commercial ELISA assay for total crustacean protein. A fourth real-time PCR assay targeting the tropomyosin gene of shrimp was compared to an ELISA assay for shrimp tropomyosin. All comparisons were carried out in two different food matrices: Manhattan clam chowder and fish sauce. PCR assays had a more broad dynamic range (0.1-106 mg/kg) as compared to ELISA (200-4000 mg/kg) and did not show matrix interference like ELISA. In cases where the ELISA assays did not have matrix interference, there was good qualitative agreement between PCR and ELISA.

Evaluation of variation within the barcode region of Cytochrome c Oxidase I (COI) for the detection of commercial Callinectes sapidus Rathbun, 1896 (blue crab) products of non-US origin.

Callinectes sapidus Rathbun, 1896 is a western Atlantic species with a disjointed natural geographic range from Massachusetts, USA to Venezuela (distribution area 1) and from Alagoas, Brazil to northern Argentina (distribution area 2). It is the only species of portunid crab commercially harvested in the continental United States but is also imported into the US from several Latin American countries, Venezuela and Mexico in particular. In the United States, crab products labeled as "blue crab" and "Product of the USA" may not legally contain other species of crab or C. sapidus not harvested in the United States. The present study documents nucleotide variation within the barcode region of cytochrome c oxidase I (COI) in 417 reference specimens of C. sapidus collected from throughout its natural range. The goal of this study is to determine if this variation can be utilized to detect mislabeled C. sapidus products sold in interstate commerce by comparing genetic signatures in reference specimens to those observed in commercial crabmeat labeled as "Product of the USA" and "Product of Venezuela." In reference specimens, we observed high levels of genetic variation in the barcode region. However, three lineages were consistently observed with significant pairwise F st values between the lineages. Lineage 1 was observed throughout the natural geographic range but predominated in the continental US and was the only lineage observed in the major crabmeat-producing states (MD, LA, VA, NC). Lineage 2 primarily occurred in the Caribbean region of distribution area 1 but was also infrequently encountered in the South Atlantic Bight region of the US coast. Finally, Lineage 3 was only observed in Brazilian waters and had the lowest haplotype and nucleotide diversity values. Lineages 1 and 2 were separated by a mean pairwise distance (p-distance) of 3.15%, whereas Lineage 3 had a mean p-distance of 2.55% and 1.35% to Lineages 1 and 2, respectively. Within lineage mean p-distances were 0.45%, 0.19%, and 0.07% for Lineages 1, 2, and 3, respectively. Among all vouchered reference specimens collected from the continental United States, Mexico, Puerto Rico, and Venezuela, we identified 22 phylogenetically informative sites that drive observed lineage divergences. Haplotypes identified from barcode COI sequences from commercial C. sapidus products labeled as originating from the US all aligned with haplotypes from Lineage 1 reference specimens and haplotypes from commercial products labeled as originating from Venezuela all aligned with Lineage 2, suggesting that these lineages may be useful for indicating whether products originate from the continental US or are imported when package labeling is in question.

Application of Multiantigen Profiling To Detect Pecan.

A problem often encountered in the detection and identification of undeclared tree nut food allergens is the lack of analytical methods. This problem is accentuated by the current trend, whereby the primary methods used to detect food allergens are antibody-based enzyme-linked immunosorbent assays (ELISAs) and the development of analyte-specific antibodies takes months. The recently developed xMAP food allergen detection assay (xMAP FADA) has the ability to generate multiantigen profiles with tree nuts, thereby providing a potential solution to this problem. The xMAP FADA includes 22 antibodies targeting peanut, soy, and nine tree nuts. The high number of antibodies to a diverse group of tree nuts and legumes and the propensity of tree nuts to cross-react have enabled the development of multiantigen profiling, whereby an analyte reacts with the various antibodies to generate a profile. Recently, a question arose regarding the possible presence of pecan dust at a manufacturer of pecan products that also stored fresh produce. The lack of suitable pecan ELISAs created an analytical challenge that was resolved using multiantigen profiling with the xMAP FADA. Pecan was detected on swab samples by using multiantigen profiling and confirmed by DNA analysis. The use of multiantigen profiling provided an analytical capability beyond what was possible with an analyte-specific analytical method.

A group-specific, quantitative real-time PCR assay for detection of crab, a crustacean shellfish allergen, in complex food matrices.

A real-time PCR assay was developed for detection of crab, a crustacean allergen, in food products. Group-specific primers and probes were developed to detect numerous species of crab. Method validation included tests of detection in complex food matrices, evaluation of commercial food products, and cross-reactivity testing on a wide variety of crustaceans. The method was able to detect several species of crab spiked into complex food matrices at levels ranging from 0.1 to 105 parts per million (weight/weight), worked equally well on different platforms, exhibited high specificity for crab over other types of crustaceans, and yielded much higher signals from commercial food products listing crab as an ingredient than from those containing other crustaceans.