Anisakids are parasitic nematodes responsible for a zoonosis caused by the ingestion of fishes infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum was found in association with gastric/intestinal illness while Hysterothylacium is considered to be not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify parasites in food products, the methods applied did not undergo through extensive validation processes, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005. Here, a comprehensive study has been undertaken to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova. Inclusivity/exclusivity trials were carried out on species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no crossreaction occurred. The LOD was determined using two independent standard curves as the minimum amount of genomic units where the reactions gave positive amplification in 10 replications. Robustness was assayed by using two different thermocyclers in three distinct laboratories. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005.
Serena, C., Alessandro, B., Enrico, A., Monica, C., Letizia, F., Antonella, C., et al. (2016). A VALIDATION PLAN FOR A COMMERCIAL KIT AIMED TO THE DETECTION OF PATHOGENIC ANISAKIDS IN FISH PRODUCTS.
A VALIDATION PLAN FOR A COMMERCIAL KIT AIMED TO THE DETECTION OF PATHOGENIC ANISAKIDS IN FISH PRODUCTS
CAFFARA, MONICA;FIORAVANTI, MARIALETIZIA;
2016
Abstract
Anisakids are parasitic nematodes responsible for a zoonosis caused by the ingestion of fishes infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum was found in association with gastric/intestinal illness while Hysterothylacium is considered to be not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify parasites in food products, the methods applied did not undergo through extensive validation processes, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005. Here, a comprehensive study has been undertaken to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova. Inclusivity/exclusivity trials were carried out on species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no crossreaction occurred. The LOD was determined using two independent standard curves as the minimum amount of genomic units where the reactions gave positive amplification in 10 replications. Robustness was assayed by using two different thermocyclers in three distinct laboratories. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
