The application of multiplex PCR method for the rapid identification of Anisakidae larvae at the species level
2018
Radosavljević, Vladimir (https://orcid.org/0000-0002-8072-4169) | Maksimović-Zorić, Jelena | Milićević, Vesna (https://orcid.org/0000-0003-1181-6307) | Veljović, Ljubiša (https://orcid.org/0000-0002-1482-3046) | Rokvić, Nikola | Pavlović, Marija (https://orcid.org/0000-0002-4854-8628) | Nešić, Ksenija (https://orcid.org/0000-0001-9255-3187)
Anisakid nematodes represent a homogeneous group of parasites whose adult stages are recovered in fish, fish-eating birds and marine mammals, while third stage larvae are often present in the body cavity and muscle of numerous fish species and also in cephalopods and crustaceans worldwide. The larvae of the genus Anisakis and Pseudoterranova, within family Anisakidae, are responsible for the human disease known as anisakiasis. Infection can be accompanied by severe allergic reactions. Major problem in the identification of Anisakis larvae in fishes is that L3 larvae of the genus Anisakis and Pseudoterranova cannot be easily differentiated morphologically. Proper identification of Anisakis species infecting host fishes is very important to both human health and fish disease diagnosis. Also, each fish species has a specific distribution area and range of hosts, thus identifying these parasites at the species level is crucial in reducing the risk for the consumer. Multiplex PCR, based on the different size of the PCR fragments amplified from a portion of the ITS region, allowed to distinguish larvae of Anisakis spp. from that of Pseudoterranova, Contracaecum and Hysterotilacium spp., and, within Anisakis spp., between A. pegreffii, A. simplex s.l. (including the hybrid genotype A. simplex/A. pegreffii), A. physeteris (including A. brevispiculata and A. paggiae) and A. typica. In this study, morphological differences of Anisakis pegreffi, Pseudoterranova decipiens and Anisakis simplex (s.s.) L3 larvae from fishes and multiplex PCR for identification of these species were evaluated. For this purposes, Anisakidae larvae preserved in ethanol were tested. Parasite DNA was isolated and used as template for the multiplex PCR. In our research, all species were successfully identified using multiplex PCR. The results confirm that the method is suitable for the identification of the Anisakidae larvae at the species level.
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