Biomphalaria mitochondrial cox

Biomphalaria mitochondrial cox 1 genotyping

Tree randomly selected Biomphalaria collected from each malacological survey site across all surveys (n = 42 Biomphalaria ) were genotyped, using PCR to amplify a

700-bp region of the Biomphalaria mitochondrial cox 1 gene according to a standard protocol [ 9]. Details of all primer sequences, reaction mixes used and PCR conditions are described in Additional file 2: Tables S1A, S1B and S1C, respectively. All Biomphalaria cox 1 PCRs included a positive control using B. pfeifferi DNA provided by the Schistosome and Snail Resource (SSR; an open access biomedical resource) [ 30] and a negative control using ddH 2 O in place of template DNA. Amplicons were visualised by running 4 µl of PCR products mixed with 1.5 µl of 5× loading buffer blue (Bioline Ltd., London, UK), stained with GelRed in a 1% agarose gel. Te PCR products were then purified using the QIAquick PCR Purification Kit (QIAGEN Ltd.) according to the manufacturer’s instructions and Sanger sequenced in the forward direction using a dilution of the LCO_1490_FW forward primer. Sequence data were visualised, trimmed and edited as needed using Geneious Prime version 2023.01 (Biomatters Ltd., Aukland, New Zealand) before being identified using the Basic Local Alignment Search Tool (BLAST) algorithm within the National Center for Biotechnology Information (NCBI) database [ 31].