DNA was extracted from swab samples using the protocol described by Franklinos et al.13 (link); however, whilst for cotton swab tips, 60 µl of PrepMan Ultra (Applied Biosystems) was added to the sample, for skin lesion tissues only 50 µl of PrepMan Ultra was used31 (link). The collected supernatant was then diluted to a 1 in 10 solution, using PCR-grade water. For negative extraction controls, sterile MW-100 swabs were used.
The Oo qPCR was conducted on a StepOnePlus™ Real-Time PCR Machine (ThermoFisher Scientific) as per Bohuski et al.32 (link) running 60 cycles (instead of 40) and using the primers Oo-rt-ITS-F and Oo-rt-ITS-R in a 20 µl reaction with 5 µl of the 1:10 template DNA31 (link). Each DNA extract was run in duplicate, and ambiguous results were repeated until consistent results were obtained: samples which generated ambiguous results after three separate runs were considered inconclusive and excluded from further analyses. Samples were considered positive if one or both of the duplicate skin swabs tested Oo qPCR positive with a cycle threshold (Ct) value of ≤ 3632 (link).
The Oo qPCR was conducted on a StepOnePlus™ Real-Time PCR Machine (ThermoFisher Scientific) as per Bohuski et al.32 (link) running 60 cycles (instead of 40) and using the primers Oo-rt-ITS-F and Oo-rt-ITS-R in a 20 µl reaction with 5 µl of the 1:10 template DNA31 (link). Each DNA extract was run in duplicate, and ambiguous results were repeated until consistent results were obtained: samples which generated ambiguous results after three separate runs were considered inconclusive and excluded from further analyses. Samples were considered positive if one or both of the duplicate skin swabs tested Oo qPCR positive with a cycle threshold (Ct) value of ≤ 3632 (link).
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