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

Gaetano Cammilleri,Chiara Drago,Giuseppe Arcoleo,Calogero Di Bella,Vincenzo Ferrantelli,Antonella Costa,Sandro Drago,Gema Del Rocio Quintero Macias,Francesco Geraci,Giuseppe Stampone,Andrea Pulvirenti

doi:10.3390/foods9010092

Abstract

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.

Highlights

The Anisakidae family includes a vast number of parasites with a worldwide distribution.The life-cycle of Anisakidae nematodes involves invertebrates, fish, cephalopods, and marine mammals so these parasites can be found in the muscles and viscera of numerous fish and cephalopod species [1,2,3,4,5].In the Mediterranean, the Anisakis spp. parasites can be found in different teleosts belonging to different ecological distributions [6]
An initial weight of 250 mg with 4 mL of extraction buffer and a 1:5 dilution after the extraction were found to be the best conditions for effective real-time detection of DNA amplification by the Loop-Mediated Isothermal Amplification (LAMP) method proposed, giving the fluorescence intensity required for detection
To reaction reduce this risk, we adopted the use system we described here can detect the concentration of Anisakis spp

Summary

Introduction

The Anisakidae family includes a vast number of parasites with a worldwide distribution.The life-cycle of Anisakidae nematodes involves invertebrates, fish, cephalopods, and marine mammals so these parasites can be found in the muscles and viscera of numerous fish and cephalopod species [1,2,3,4,5].In the Mediterranean, the Anisakis spp. parasites can be found in different teleosts belonging to different ecological distributions [6]. The Anisakidae family includes a vast number of parasites with a worldwide distribution. The life-cycle of Anisakidae nematodes involves invertebrates, fish, cephalopods, and marine mammals so these parasites can be found in the muscles and viscera of numerous fish and cephalopod species [1,2,3,4,5]. In the Mediterranean, the Anisakis spp. parasites can be found in different teleosts belonging to different ecological distributions [6]. The fish species mainly involved in the life cycle of Anisakis belong to the pelagic, benthopelagic, and benthodemersal domains. Foods 2020, 9, 92 occurring in pelagic, benthopelagic, and demersal species of the Gadidae, Merlucciidae, Scombridae, Carangidae, and Trichiuridae families [2,7,8]. Anisakis spp. parasites are marine nematodes of health interest because of their high zoonotic potential, being responsible for a human disease called

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