Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis)

Autor: Loïc Durand, Catherine Cazeaux, Marco Lalle, Jitender P. Dubey, Stéphanie La Carbona, Dominique Aubert, Isabelle Villena, Alain Geffard, Loïc Favennec
Přispěvatelé: ACTALIA [Villers-Bocage], Istituto Superiore di Sanita [Rome], Epidémiosurveillance de protozooses à transmission alimentaire et vectorielle (ESCAPE), Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Université de Reims Champagne-Ardenne (URCA), Centre Hospitalier Universitaire de Reims (CHU Reims), Animal Parasitic Diseases Laboratory [Beltsville, USA], United States Department of Agriculture (USDA)-USDA Agricultural Research Service [Beltsville, Maryland], USDA-ARS : Agricultural Research Service-USDA-ARS : Agricultural Research Service, Stress Environnementaux et BIOsurveillance des milieux aquatiques (SEBIO), Université Le Havre Normandie (ULH), Normandie Université (NU)-Normandie Université (NU)-Université de Reims Champagne-Ardenne (URCA)-SFR Condorcet, Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Centre National de la Recherche Scientifique (CNRS)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Centre National de la Recherche Scientifique (CNRS)-Institut National de l'Environnement Industriel et des Risques (INERIS)
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: Food Microbiology
Food Microbiology, Elsevier, 2022, 102, pp.103870. ⟨10.1016/j.fm.2021.103870⟩
ISSN: 0740-0020
1095-9998
Popis: The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.
Databáze: OpenAIRE
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