Trichinella larvae

Murakami, Masaki, Tokiwa, Toshihiro, Sugiyama, Hiromu, Shiroyama, Mitsuko, Morishima, Yasuyuki, Watanabe, Sota, Sasamori, Takato, Kondo, Mami, Mano, Tsutomu & Tsuruga, Hifumi, 2023, Trichinella T 9 in wild bears in Japan: Prevalence, species / genotype identification, and public health implications, International Journal for Parasitology: Parasites and Wildlife 21, pp. 264-268 : 265-266

2.3. Molecular identification of Trichinella larvae

DNA samples were extracted from a pool of larvae collected from each animal (4– 100 larvae /head) using a QIAamp DNA Mini Kit ( Qiagen , Hilden, Germany). Two sets of primer pairs (5′-

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CACCCAGAAGTATACATCC-3′ and 5′-GTAATAATAGGTCTAGGGAGG-3′ for cytochrome c oxidase subunit I gene, cox1, and 5′-CAATT- GAAAACCGGTGAG-3′ and 5′-ATCACTCAACATTAACCG-3′ for the nuclear internal transcribed spacer 2 region, ITS2) were used to identify the Trichinella species following the methods described in a previous report ( Kanai et al., 2006). The PCR products were visualised using electrophoresis on a 1.5% agarose gel and subjected to direct sequencing at Eurofins Genomics, Inc. (Tokyo, Japan). Multiple sequence alignments were performed using MAFFT ver. 7.505 with the option Q–INS–I ( Katoh and Standley, 2016). The sequences obtained were compared to those available in the International Nucleotide Sequence Database at NCBI using the BLASTN program (http://blast.ncbi.nlm.nih.gov/Blast. cgi).