Syngamus trachea

Meister, Seraina L., Wenker, Christian, Wyss, Fabia, Zühlke, Irene, In, Veiga, es Berenguer & Basso, Walter U., 2022, Syngamus trachea in free-ranging white stork (Ciconia ciconia) nestlings in Switzerland, International Journal for Parasitology: Parasites and Wildlife 18, pp. 76-81 : 77

2.6.1. Syngamus trachea molecular analysis

A PCR targeting the mitochondrial cytochrome c oxidase subunit I gene (cox-1) was performed ( Casiraghi et al., 2001). One primer set (Microsynth AG, Balgach, Switzerland) was used at a 100 μM working solution. Per tube, 0.25 μl of each primer (COIintF and COIintR) were mixed with 12.5 μl of a commercial master mix (QIAGEN Multiplex PCR Kit, Hombrechtikon, Switzerland), 9.5 μl ddH 20 and 2.5 μl of DNA template to a total volume of 25 μl. The PCR amplification was performed under the following conditions: 95 ◦ C for 15’ (for activation of HotStarTaq DNA Polymerase), followed by 40 cycles of denaturation at 94 ◦ C for 45 ′′, annealing at 52 ◦ C for 45 ′′ and extension at 72 ◦ C for 90 ′′ with a final extension at 72 ◦ C for 10’. A known positive DNA control and a negative control (ddH 2 O) were used for each run. Amplification products were resolved by gel electrophoresis in 1.5% agarose gels containing ethidium bromide for 35 min at 120 V. DNA was recovered from the PCR products using the Zymo Research Purification Kit (DNA Clean & ConcentratorTM-5, Zymo Research Europe GmbH, Lucerne, Switzerland). Subsequently, a bidirectional sequencing of the obtained amplicons was performed with the same primers used in the PCR reaction (Microsynth AG, Balgach, Switzerland), followed by comparison with sequences on GenBank® using the Nucleotide BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The obtained DNA sequences were deposited in GenBank® after trimming the primer-binding regions (accession no. OM831258 - OM831262).