2.3 DNA EXTRACTION AND PATHOGEN DETECTION
To detect endoparasites the gut of each specimen has been extracted
using tweezers sterilized using a 3% bleach solution and 70% ethanol
in between dissections to avoid contamination between individuals.
Subsequently, the genomic DNA was extracted from gut samples by using
Qiagen© DNeasy® PowerSoil Pro Kit (Qiagen, Hilden, Germany) following
the manufacturer’s protocol. Target DNA fragments, exclusive for the
investigated endoparasites, have been amplified through PCR assays.
Specific primer pairs were adopted to amplify sequences belonging to 18S
region (SSU) for trypanosomatids detection, 16S region (SSU) for
microsporidia detection and sequences that include part of 5.8S, section
of ITS2 and part of 28S section (SSU) for Apicystis bombidetection (see Table 1 for further information and references).
WonderTaq® DNA polymerase was used for amplification following the
manufacturer instruction with 5 µL of DNA template. The thermal profile
used to perform PCR was the same for all three primer pairs and was
organized in 10 pre-amplification cycles of 30s at 94°C, 30s at 60°C and
45s at 72°C followed by 30 amplification cycles of 30s at 94°C, 30s at
57°C and 45s at 72°C (Graystock et al., 2020).
Capillary electrophoresis was performed with Qiagen© QIAxcel® Advanced
System (Qiagen, Hilden Germany), using the Qiagen© QIAxcel® DNA
High-Resolution screening kit (Qiagen, Hilden Germany) in order to
visualize expected band presence and size related to each parasite (see
Table 1). A pool of positive samples for each endoparasite is depicted
in Figure S1.
[ TABLE 1]