Multiplex PCR for the identification of Anisakis simplex sensu stricto, Anisakis pegreffii and the other anisakid nematodes

Anisakis (Nematoda: Ascaridoidea) from Indonesia

“…They were compared manually with the original chromatograms, identified via GenBank and aligned with a previously characterized sequence (GenBank) data of Ansiakis spp., and with 125 previously identified Anisakis sequences from Indonesia (see , Koinari et al 2013, Kuhn et al 2013, Anshary et al 2014, Kleinertz et al 2014a) using ClustalW (v. 1.83) multiple sequence alignments (settings: full multiple alignment, gap penalties default) (Thompson et al 1994). They were then aligned with sequences from other Anisakis species from GenBank for species and genotype identification as follows: A. typica (s.s.) from the spinner dolphin Stenella longirostris from Brazil (AY826724; see Nadler et al 2005), A. simplex C, now A. berlandi from Mirounga angustirostris from California (AY 821739; see Nadler et al 2005), A. pegreffii and A. physeteris from mackerels from Japan (AB277823 and AB277821 respectively; see Umehara et al 2008), and Anisakis sp. HC-2005 from Hoplostethus cadenati from the African shelf (EU718474; see Kijewska et al 2009).…”

Türkiye’nin Erzurum İlinde İnsan Tüketimi İçin Satılan İstavrit Balıklarında (Trachurus trachurus) Anisakis pegreffii’nin Moleküler Tanısı

“…All six parasites showed three bands of 250, 300, 370 bp which were specific to A. pegreffii. Researchers obtained the same banding pattern for A. pegreffii in different studies 5,7,14,16,22,25 . Umehara et al 5 and Fang et al 6 developed speciesspecific primers from the ITS region of rDNA and used in multiplex PCR for the rapid identification of anisakid nematodes.…”

“…Universal primers A (5′-gtcgaattcgtaggtgaacctgcggaa ggatca-3′) and B (5′-gccggatccgaatcctggttagtttcttttcct-3′) were used for the amplification of rDNA (ITS1, 5.8 subunit rRNA and ITS2) 5,7,[14][15][16] . PCR was carried out in a final volume of 50 μl, containing 23.75 μl DNase, RNase free steril distilled water (Biobasic, Inc), 5 μl 10X PCR buffer, 6 μl 25 mM MgCl 2 , 5 μl 1 mM dNTP mix, 2.5 μl of each primer (50 pmol), 5 μl of template DNA, and 0.25 μl of TaqDNA polymerase (1.25 IU) (MBI, Fermentas).…”

“…APE1 (5′-gagcagcagcttaaggcagaggc-3′) and B (5′-gccg gatccgaatcctggttagtttcttttcct-3′) primer pair was specific to A. pegreffii and used for the partial amplification of 18S-28S rRNA gene 5 . PCR mix and PCR conditions were same as above.…”

Anisakis spp. induced granulomatous dermatitis in a harbour porpoise Phocoena phocoena and a bottlenose dolphin Tursiops truncatus

“…The first PCR targeted the nematodes' partial 18S gene sequence using the previously published primers Nem_S_F (forward) and Nem_18S_R (reverse) (Floyd et al 2005). The second PCR targeted the 18S-28S region (including ITS1, 5.8S and ITS2), using the previously published primers nemspec 18SF (forward) and NC2 (reverse) (Umehara et al 2008). The thermal profile of both PCRs consisted of a denaturation step of 94°C for 5 min, 45 cycles of 94°C for 30 s, 54 or 52°C, respectively, for 30 s and 72°C for 1 min, and a final extension step at 72°C for 10 min, and was carried out in a T100 Thermal Cycler (Bio-rad).…”