Anisakis pegreffii: A quantitative fluorescence PCR assay for detection in situ

[Display omitted] ► A real-time PCR method for the detection in situ of Anisakis pegreffii was developed. ► The ITS-2 of rDNA of A. pegreffii was used as molecular marker. ► The assay was capable of detecting 1/3 L3 in 30mg of fish tissue. To facilitate improved diagnosis and detection of the third stage larva (L3) of Anisakis pegreffii from the Minnan-Taiwan bank fishing ground in Taiwan Strait, a real-time PCR method for the detection in situ and differentiation was developed to amplify a region of the second internal transcribed spacer (ITS-2) of this parasite. The real-time PCR assay was capable of detecting 1/3 of a single L3 in 30mg of marine fish tissue, and also exhibited a high level of specificity for A. pegreffii, no fluorescence signals were observed in other five major larval anisakid species found in commercial marine fishes caught in this fishing ground.