Evaluation of a Real-Time Polymerase Chain Reaction (PCR) Assay for Detection of Anisakis simplex Parasite as a Food-Borne Allergen Source in Seafood Products

Abstract

Anisakis simplex has been recognized as an important cause of disease in humans and as a food-borne allergen source. Actually, this food-borne parasite was recently identified as an emerging food safety risk. An A. simplex -specific primer-probe system based on a real-time polymerase chain reaction (PCR) detection assay has been successfully optimized and validated with seafood samples. In addition, a DNA extraction procedure has been optimized to detect the presence of the nematode in food samples. The assay is a very reliable, specific, and sensitive methodology to detect the presence of traces of this parasite in seafood products, including highly processed samples. As a result, 13 sequences of cytochrome c oxidase II gene were obtained and scrutinized to calculate intra- and interspecific variabilities of 0 and 35-67%, respectively. Finally, an efficiency of 2.07 +/- 0.14 of the assay was calculated, and a limit of detection of 40 ppm parasite in 25 g of sample was also optimized. Actually, the presence of this parasite in several seafood products has been demonstrated, enforcing the necessity of a design for a good manufacturing practice protocol for the processing industry to minimize the presence of this parasite as a food-borne allergen source in seafood products.

Putative hybrids between two Anisakis cryptic species: Molecular genotyping using High Resolution Melting

“…In fact, this technique has been used by now only for rapid discrimination of Brugia malayi and Brugia pahangi (Areekit et al, 2009), in population studies of Fascioloides magna (Radvansky et al, 2011) and for human hookworms discrimination (Ngui et al, 2012). Real time PCR approaches have been used for the detection of anisakid nematodes (Zhang et al, 2010), to reveal residues in food products (Mossali et al, 2010) as well as food-borne allergen sources in seafood products (Lopez and Pardo, 2010), but HRM approach is still poorly used, despite its great versatility as a screening approach or mutation scanning analysis. Its use has confirmed a third genetic profile for the putative hybrids, but it does not definitively assess the hybrid status to these specimens.…”

Fluorescent magnetic bead-based mast cell biosensor for electrochemical detection of allergens in foodstuffs

“…There currently exist a number of technical approaches for identifying the presence of allergic proteins in food,includingmass spectrometry (Carrera et al,2012),real-time polymerase chainreaction (RT-PCR) (Lopez and Pardo, 2010;Sun et al, 2009), and enzyme-linked immunosorbent assay (ELISA) (Fu and Maks, 2013;Zhang et al, 2014).Though these tests are highly specific and sensitive, their lengthy analysis time poses an obvious inconvenience to practical applications.Other possible methods for specific food allergen detection are based on the measurement of IgE-induced degranulation in vitro (Eccleston et al,1973).For example, the rat basophilic leukemia (RBL-2H3) cell line possesses FcR, which can selectively bind murine IgE antibodies so that subsequent encounters with an allergen will cross-link the FcRI-IgE complex on the surface of mast cells (Curtis et al,2008). This triggers a sequence of intracellular events, including cellular degranulation and the release of chemical mediators such as histamine, serotonin, and β-hexosaminidase (Brockow, 2013;Corry and Kheradmand, 1999).…”

A quantitative sandwich ELISA for the detection of Anisakis simplex protein in seafood

“…Therefore, food safety authorities and seafood manufacturers are likewise interested in improved methods for the detection of Anisakis in foods. So far, only visual inspection methods of more or less intact worms [20,21] or PCR methods for the determination of parasite DNA [22,23] in fish products are available. However, there are at present no validated methods for the quantification of Anisakis protein in processed fish products and seafood.…”

“…The applicability of the assay for the quantification of Anisakis proteins in processed foods was not reported. In contrast, several PCR-based methods been developed that are intended for the quantification of parasite residues in food products [22,23]. PCR-and ELISAbased methods can be regarded as complementary techniques with respect to allergen detection.…”