Detection of Bsal infection
The genomic DNA for skin swabs was extracted using the PreMan Ultra
(Applied Biosystems) (Boyle et al., 2004) and Qiagen DNeasy Blood and
Tissue Kit, following animal tissue part in the modified manufacturers’
protocols (Kosch & Summers, 2012). The environmental DNA for filters
was extracted using Qiagen DNeasy PowerSoil Kit.
We used nested PCR to detect Bsal on DNA extracts from skin swabs
(Gaertner et al., 2009; Zhu et al., 2014). For the first amplification,
the primers ITS1f and ITS4 were used which specifically combined the 18S
and 28S rRNA genes. The PCR amplification conditions,
4 min at 94°C, followed by 30 cycles
of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C and a final 10 min at
72°C were implemented. For the second amplification, the specific
primers (STerF and STerR) for Bsal were used to amplify a
fragment gene of the ITS-5.8S rRNA region (Martel et al., 2013). The
conditions of PCR amplification included 4 min at 94°C, followed by 30
cycles of 30 s at 94°C, 60°C for 30s, and 1 min at 72°C and a final 10
min at 72°C. PCR amplification products were visualized by 1.5% agarose
gel electrophoresis.
Environmental samples were tested for Bsal using the RT-PCR
method described by Blooi et al . (2013, 2016). We used a cycle of
an initial step at 95°C for 10 min, followed by 50 cycles of 95°C for 10
s and 60°C for 1 min on Light Cycler® 480 System (Roche, Mannheim,
Germany). Each water sample was run in duplicate.
We used the synthetic DNA sequences of Bsal as positive controls
(Pisces Molecular, Boulder, Colorado, USA) and two negative controls in
each plate. If nested PCR and RT-PCR products yielded positive results,
Sanger sequencing was conducted to verify if the amplified DNA fragments
were indeed associated with Bsal .