An economical and combined method for rapid and efficient isolation of fungal DNA

Abstract

DNA isolation is a crucial step of conducting genetic studies in any organism. However, this process is quite difficult when studying fungi because of the need to damage the fungal cell walls of specific structures. In this study, we developed a method for the rapid and efficient isolation of fungal DNA based on simultaneous mechanical and enzymatic cell wall degradation. There are several typical modifications of the standard phenol-chloroform DNA extraction method. This method can be modified to degrade the fungal cell wall. The first step of the presented DNA extraction included manual homogenization in modified lysis buffer. Next, enzymatic digestion using 2 enzymes was conducted, including lyticase and proteinase K. To carefully select the most favorable conditions, we developed an economical, rapid, and reliable method for fungal DNA extraction that ensures both high efficiency and proper purity, which are essential for further analyses.

Optimizing a real-time PCR assay for rapid detection of Candida auris in nasal and axillary/groin samples

“…Better DNA recovery after lyticase treatment was reported by Karakousis et al [16]. Results from the study by Lech et al showed that double-digestion with two enzymes, lyticase and proteinase K, improved the DNA isolation procedure and shortened the time of incubation with various enzymes [17]. Therefore, for enzymatic cell disruption, the use of two enzymes may be required to achieve an optimal result.…”

Evaluation of a modified sampling method for molecular analysis of air microflora

“…For DNA isolation, the standard multi-phased phenol-chloroform method proposed by Byrd et al (1990) was used with modifications. The phenol-chloroform method provides satisfactory recovery of DNA from environments such as indoor air, which contain low DNA concentration (Lech et al, 2014). Prepared samples collected from the air were flooded with 400 mL optimized lysis buffer (100 mM EDTA; 100 mM Tris-HCl, pH 8, 0.5 M NaCl; 3% sodium dodecyl sulfate) with the addition of lyticase enzyme (400 U/sample).…”