Biomphalaria infections With

Conformation of Biomphalaria infections With other Trematoda species

To identify Trematoda species other than S. mansoni infecting Biomphalaria , we subjected all samples that successfully amplified only Biomphalaria and Trematoda ITS loci to a secondary singleplex PCR, again to amplify only the Biomphalaria ITS and Trematoda ITS loci. To do this, the molecular xenomonitoring PCR was repeated, but with replacement of the S. mansoni ND 5 forward and reverse primers with ddH 2 O. Amplicons were visualised in the same manner as for the multiplex molecular xenomonitoring PCRs and the approximately 1005-bp Trematoda ITS gel band was excised using a fresh scalpel. Excised gel bands were purified using the QIAquick Gel purification kit (Qiagen Ltd.) according to manufacturer’s instructions and were then purified as described above prior to Sanger sequencing in the forward direction using a dilution of the ETTS2 forward primer. Sequence data were visualised, trimmed and edited as needed using Geneious Prime version 2023.01 (Biomatters, Ltd.) before being identified using the BLAST algorithm within the NCBI database [ 31].