Rapid identification of Anisakis spp. larvae is very interesting for fish food industry. Nevertheless, biomolecular techniques are labour-intensive and time-consuming, and they require technical know-how and expensive equipment. Loop-mediated isothermal amplification (LAMP) is a relatively new technique and it proposes significant advantages in terms of ease and speed; moreover, it has already been developed and extensively used as a diagnostic tool for some pathogenic bacteria sush as Listeria monocytogenes and parasites like Toxoplasma gondii [1, 2]. In this study, a specific primer was designed and a fluorescence-based real-time LAMP assay was studied, providing an alternative and primarily screening approach for the identification of the genus Anisakis. The primer developed was tested directly against Anisakis spp. and other non-pathogenic Anisakidae larvae (Hysterothylacium fabri). Subsequently, the kit was tested against homogenized fish muscle voluntarily contaminated with a decreasing number of larvae, previously isolated and identified to genus level. Finally, baby food samples (80g) contaminated with one, two and three larvae were also tested. The specificity of the kit developed to amplify DNA sequences belonging to Anisakis spp. was confirmed as larvae belonging to Anisakis simplex sensu strictu and A. pegreffii were amplified and recognized by the kit, while Hysterothylacium fabri, a non zoonotic parasite morphologically similar to Anisakis spp. and belonging to the same family, was not recognized. The tests performed using mixed fish muscle in order to evaluate the sensitivity of the kit resulted to be in agreement with the biomolecular tecniques actually used [3, 4] with a sensitivity equal to 20 larvae/kg of product. Comparable sensitivity was also confirmed with the test perfomed using baby food [5]. LAMP tecnique applied could have promising practical outcomes as it is able to produce good identification results in short times and with low costs. Important advantages could be especially produced if applied to the periodical control of fish-based products (like baby food) or to quality control of extensive productions before commercialization.
Loop-mediated isothermal amplification (LAMP) for the detection of Anisakis simplex / E. Tirloni, S. Stella, S. Drago, G. Stampone, C. Bernard - In: Convegno SISVET[s.l] : SISVET, 2017. - ISBN 9788890909245. - pp. 201-201 (( Intervento presentato al 71. convegno Convegno SISVET tenutosi a Napoli nel 2017.
Loop-mediated isothermal amplification (LAMP) for the detection of Anisakis simplex
E. Tirloni
;S. Stella;
2017
Abstract
Rapid identification of Anisakis spp. larvae is very interesting for fish food industry. Nevertheless, biomolecular techniques are labour-intensive and time-consuming, and they require technical know-how and expensive equipment. Loop-mediated isothermal amplification (LAMP) is a relatively new technique and it proposes significant advantages in terms of ease and speed; moreover, it has already been developed and extensively used as a diagnostic tool for some pathogenic bacteria sush as Listeria monocytogenes and parasites like Toxoplasma gondii [1, 2]. In this study, a specific primer was designed and a fluorescence-based real-time LAMP assay was studied, providing an alternative and primarily screening approach for the identification of the genus Anisakis. The primer developed was tested directly against Anisakis spp. and other non-pathogenic Anisakidae larvae (Hysterothylacium fabri). Subsequently, the kit was tested against homogenized fish muscle voluntarily contaminated with a decreasing number of larvae, previously isolated and identified to genus level. Finally, baby food samples (80g) contaminated with one, two and three larvae were also tested. The specificity of the kit developed to amplify DNA sequences belonging to Anisakis spp. was confirmed as larvae belonging to Anisakis simplex sensu strictu and A. pegreffii were amplified and recognized by the kit, while Hysterothylacium fabri, a non zoonotic parasite morphologically similar to Anisakis spp. and belonging to the same family, was not recognized. The tests performed using mixed fish muscle in order to evaluate the sensitivity of the kit resulted to be in agreement with the biomolecular tecniques actually used [3, 4] with a sensitivity equal to 20 larvae/kg of product. Comparable sensitivity was also confirmed with the test perfomed using baby food [5]. LAMP tecnique applied could have promising practical outcomes as it is able to produce good identification results in short times and with low costs. Important advantages could be especially produced if applied to the periodical control of fish-based products (like baby food) or to quality control of extensive productions before commercialization.File | Dimensione | Formato | |
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