“…PCR amplification of the rDNA sequence by the universal fungal primers, followed by DNA sequencing and BLAST search against the GenBank database, have been used for the definitive identification of P. insidiosum for decades (Badenoch et al, 2001). The multistep procedure, limited efficiency, and long turnaround time of such sequence homology-based analysis can be overcome by using the P. insidiosum-specific primers, alternative target genes (i.e., exo1 and cox2), and advanced DNA amplification techniques, such as nested PCR (Grooters and Gee, 2002;Thongsri et al, 2013), singleround species-specific PCR (Vanittanakom et al, 2004;Keeratijarut et al, 2014), real-time PCR (Keeratijarut et al, 2015;Worasilchai et al, 2018b), thermophilic helicase DNA amplification (Worasilchai et al, 2018a), and multiplex PCR (Rujirawat et al, 2017;Weiblen et al, 2019;Kulandai et al, 2020). A primary concern regarding the molecular diagnosis for pythiosis is the lack of costly equipment in remote areas, where the disease is prevalent.…”