Detection and identification of anisakids in seafood by fragment length polymorphism analysis and PCR–RFLP of ITS-1 region

Abstract

Anisakids are a group of widely distributed nematodes which have acquired high social relevance due to their involvement in foodborne infections caused by consumption of raw or undercooked seafood. The current study has been focused on the development of a rapid and sensitive method for the detection of anisakids in seafood based on the polymerase chain reaction. The molecular marker studied has been the Internal Transcribed Spacer 1. The analytical strategy consists of two steps. The first step is based on specific primers and the length polymorphism of the amplified PCR product. This first probe allows detecting the presence of anisakids in fish, and in some cases determining the particular anisakid species. When the first step is not conclusive as to the exact anisakid species present in the sample, the restriction fragment length polymorphism analysis was carried out, allowing to assign the resulting profile to different anisakid species. The main novelty of this work lies is in the fact that it allows the simultaneous detection and identification of the most important anisakid species present in fish products. Also, two methodological alternatives were developed to adapt this method to different laboratories depending on the availability of equipment, one performed with simple instrumentation (agarose gels), and the other with more sophisticated instrumentation (Genetic Analyzer). Both can be applied to all kinds of processed products, including those undergoing intensive processes of transformation, as for instance canned foods. The proposed methodologies are rapid, robust, highly sensitive and readily adaptable in routine molecular diagnostic laboratories.