Quantitative SYBR Green qPCR technique for the detection of the nematode parasite Anisakis in commercial fish-derived food

Godínez-González, Carla; Roca-Geronès, Xavier; Cancino-Faure, Beatriz; Montoliu, Isabel; Fisa, Roser

doi:10.1016/j.ijfoodmicro.2017.05.012

Cited by 13 publications

(16 citation statements)

References 41 publications

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“…The high prevalence and the intensity of invasion of Anisakis in hake and mackerel are known; therefore, these fish species considered to be “high-risk” for consumers [ 48 , 52 , 53 ]. The presence of Anisakis antigen in surimi sticks, as already mentioned, was found in our study and in other investigations [ 29 , 47 ]. The manufacturer of surimi sticks examined in the present study did not specify the species of fish used to make this product.…”

Section: Discussionsupporting

confidence: 92%

“…To date, several different immunoenzymatic [ 16 , 21 , 22 , 23 ], molecular [ 24 , 25 , 26 , 27 , 28 , 29 , 30 ], and mass spectrometry [ 31 , 32 ] methods have been developed for the detection of A simplex in processed fish products. Nevertheless, only a few assays have been validated using samples exposed to high temperatures similar to those used in food technological processes [ 30 , 33 ].…”

Section: Discussionmentioning

confidence: 99%

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Development and Application of Novel Chemiluminescence Immunoassays for Highly Sensitive Detection of Anisakis simplex Proteins in Thermally Processed Seafood

et al. 2020

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The third-stage larvae (L3) of Anisakis simplex are the most important source of hidden allergens in seafood products. However, there exist no commercial methods for detecting Anisakis proteins in food. Furthermore, only a few methods have been validated for the detection of A. simplex in thermally processed food. The aims of our study are (i) the development and validation of high-sensitivity chemiluminescent (CL) immunoassays for the detection of A. simplex proteins in processed seafood, (ii) and A. simplex antigen detection in common seafood products from Polish markets. We developed and validated CL sandwich ELISA (S-ELISA) and CL competitive ELISA (C-ELISA) methods for A. simplex proteins detection in food, with respective detection limits of 0.5 and 5 ng/mL. The usefulness of the assays for detecting A. simplex proteins in highly processed food was evaluated by examination of autoclaved canned fish spiked with A. simplex larvae (1–8 larvae/200 g). Commercial real-time PCR was unable to detect A. simplex in autoclaved samples at all levels of enrichment with Anisakis larvae. CL-S-ELISA was used to test various types of seafood products from Polish markets. Among all tested products (n = 259), 28% were positive. A. simplex antigens were found mostly (n = 39) in smoked fish products: mackerel, herring, cod, and hake. Other positive samples were found in marinated herrings, canned cod livers, canned mackerels, and surimi sticks. In tuna, Atlantic argentine, anchovy, sardine, sprat, and squid products, A. simplex antigens were not detected. This study provides novel effective tools for the detection of A. simplex proteins in processed food and highlights the potential allergic hazards for Anisakis-sensitized Polish consumers of seafood.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Development and Application of Novel Chemiluminescence Immunoassays for Highly Sensitive Detection of Anisakis simplex Proteins in Thermally Processed Seafood

et al. 2020

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“…High target copy number may explain the high sensitivity of the test we deployed. The mitochondrial gene COI, has been widely used to detect the presence of nematode parasites in commercially important fish species (Godínez-González et al, 2017;Herrero, Vieites, & Espiñeira, 2011;Paoletti et al, 2018;Santos et al, 2006). Many studies rely on qPCR or Real Time PCR for fish pathogens detection, but such approaches are challenging and expensive to deploy in the field (Chlo Suzanne Hutchins et al 2018;Trujillo-González et al 2019).…”

Section: Discussionmentioning

confidence: 99%

“…In other studies quantitative qPCR has been developed to detect Anisakis sp. in experimentally prepared fish-derived food products (Godínez-González et al 2017, Mossali et al 2010. A non-lethal qPCR based eDNA approach has also been optimized to detect the cestode Schistocephalus solidus in samples taken by needle from the intra-peritoneal cavity of a fish (Chloé Suzanne ).…”

Section: Introductionmentioning

confidence: 99%

In-situnon-lethal rapid test to accurately detect the presence of the nematode parasite,Anguillicoloides crassus, in European eel,Anguilla anguilla

Noia

Poole

Kaufmann

et al. 2020

Preprint

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Anguillicoloides crassus is an invasive nematode parasite of the European eel, Anguilla anguilla, and one of the primary drivers of eel population collapse. The presence of the parasite has been shown to impact many features of eel physiology and life history. Early detection of the parasite is vital to limit the spread of A. crassus. However, until recently, accurate diagnosis of infection could only be achieved via terminal dissection. To support A. anguilla fisheries management in the context of A. crassus we developed a rapid non-lethal and non-invasive environmental DNA method to detect the presence of the parasite in the swim bladder. Screening of 131 wild eels was undertaken between 2017 and 2019 Ireland and UK to validate the procedure. DNA extractions and PCR were conducted using both a Qiagen Stool kit at Glasgow University and in situ using Whatman qualitative filter paper No. 1 and a miniPCR DNA Discovery System™.Primers were specifically designed from the cytochrome oxidase mtDNA gene region. In situ extraction and amplification takes approx. 3h for up to 16 individuals with higher specificity and sensitivity compare to the laboratory Qiagen kit extraction. The local diagnostic procedure demonstrated Positive Predictive Values at 96% and Negative Predictive Values at 87%. Our method will be a powerful tool in the hands of fisheries managers to help protect this iconic but critically endangered species. It will allow a non-invasive monitoring of the A. crassus dispersion across the European waters.

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“…In this case, molecular biology methods are valuable tools in the detection of Anisakis spp. nematodes in processed seafood [24,[27][28][29][30]. Several studies have shown that immunological and molecular methodologies yield comparable results concerning the detection of allergens in processed foods as sensitive and specific tools [31][32][33].…”

Section: Introductionmentioning

confidence: 99%

Validation of a Commercial Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Anisakis spp. DNA in Processed Fish Products

Cammilleri

Ferrantelli

Pulvirenti

et al. 2020

Foods

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Parasites belonging to the Anisakis genera are organisms of interest for human health because they are responsible for the Anisakiasis zoonosis, caused by the ingestion of raw or undercooked fish. Furthermore, several authors have reported this parasite to be a relevant inducer of acute or chronic allergic diseases. In this work, a rapid commercial system based on Loop-Mediated Isothermal Amplification (LAMP) was optimised and validated for the sensitive and rapid detection of Anisakis spp. DNA in processed fish products. The specificity and sensitivity of the LAMP assay for processed fish samples experimentally infected with Anisakis spp. larvae and DNA were determined. The LAMP system proposed in this study was able to give positive amplification for all the processed fish samples artificially contaminated with Anisakis spp., giving sensitivity values equal to 100%. Specificity tests provided no amplification for the Contracaecum, Pseudoterranova, or Hysterothylacium genera and uninfected samples. The limit of detection (LOD) of the LAMP assay proposed was 102 times lower than the real-time PCR method compared. To the best of our knowledge, this is the first report regarding the application of the LAMP assay for the detection of Anisakis spp. in processed fish products. The results obtained indicate that the LAMP assay validated in this work could be a reliable, easy-to-use, and convenient tool for the rapid detection of Anisakis DNA in fish product inspection.

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Quantitative SYBR Green qPCR technique for the detection of the nematode parasite Anisakis in commercial fish-derived food

Cited by 13 publications

References 41 publications

Development and Application of Novel Chemiluminescence Immunoassays for Highly Sensitive Detection of Anisakis simplex Proteins in Thermally Processed Seafood

Development and Application of Novel Chemiluminescence Immunoassays for Highly Sensitive Detection of Anisakis simplex Proteins in Thermally Processed Seafood

In-situnon-lethal rapid test to accurately detect the presence of the nematode parasite,Anguillicoloides crassus, in European eel,Anguilla anguilla

Validation of a Commercial Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Anisakis spp. DNA in Processed Fish Products

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