Application of the PCR-sequence-specific primers for the discrimination among larval Anisakis simplex complex

The fish-borne zoonotic nematode, Anisakis simplex, is not a single specie but a complex composed of three sibling species, A. simplex sensu stricto, Anisakis pegreffii, and A. simplex C. Discrimination among these sibling species have been performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism or sequencing analysis of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). In the present study, PCR-sequence-specific primers were applied for the discrimination of these parasites. Based on the sequence differences among A. simplex complex at the ITS region of rDNA, forward primer ASF1 that is expected to be specific to A. pegreffii was newly developed. The specific fragment was amplified in only A. pegreffii by PCR with ASF1, but not quite in A. simplex s. str., Anisakis physeteris, Pseudoterranova decipiens, and Contracaecum osculatum. This result suggests that PCR using the forward primer ASF1 might be applicable for the discrimination of A. simplex complex.