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

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.

One-step colorimetric isothermal detection of COVID-19 with AI-assisted automated result analysis: A platform model for future emerging point-of-care RNA/DNA disease diagnosis

“…Regarding the limitation of our assay, fundamentally, the introduction of LAMP-XO has served as a basis for the advent of the LG indicator that offers a different end result color from its parent dyes. Under a certain condition, though, the color development of the lavender green system is adversely affected by the presence of mineral oil often added into the LAMP reaction to prevent its content from evaporating when the choice of heat source is not itemized with a heated lid control [ [47] , [48] , [49] , [50] , [51] ]. Thus, the chemical nature of this composite indicator still needs to be investigated in order to utilize it to the fullest potential.…”

Negligible risk of zoonotic anisakid nematodes in farmed fish from European mariculture, 2016 to 2018

“…When enhanced with the use of feasible and reliable parasite detection methods for the processed fish products (homogenised fish product, etc.) [ 36 ], it might additionally aid to maintain the likelihood of zoonotic parasite occurrence in all farmed fish at a very low level.…”

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

“…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 ].…”