Evaluation of different DNA extraction methods based on steel‐bullet beating for molecular diagnosis of onychomycosis

Abstract

Background Considering increased trends toward molecular methods for detection/identification of fungi causing onychomycosis, the aim of this study is comparison three DNA extraction methods based on steel‐bullet beating to extract DNA from nail. Methods Ex ‐vivo onychomycosis model was developed using bovine hoof with Candida albicans and Aspergillus flavus. For two models, total DNA was extracted using the three different methods. In method 1, the extraction and purification were performed by steel‐bullet beating and phenol chloroform protocol, respectively. In method 2, a freezing step were applied before beating. The purification step in method 3 was carried out using a commercial kit, although DNA extraction was done similarly to method 1 in that approach. To evaluate the efficacy of each method, the extracted genomic DNA was amplified with Polymerase Chain Reaction (PCR) using Internal Transcribed Spacer (ITS) regions. Moreover, 50 nail samples were evaluated for onychomycosis using direct microscopy examination as well as PCR in order to evaluate the diagnostic efficiency of the optimal DNA extraction method. Results Regarding the desirable quality of the extracted DNA, cost effectiveness, and simplicity, method 1 could be used to extract DNA effectively. Additionally, the obtained data showed that PCR had a higher detection rate of fungal agents in the nail samples than direct microscopic examination. Conclusions This study demonstrated that the mechanical disruption of the cell wall by steel‐bullet beating is a useful and practical method to improve the quantity and quality of fungal DNA thorough the extraction process.

Candida spp. DNA Extraction in the Age of Molecular Diagnosis

“…A more cost-effective alternative to bead beating could arise: steel-bullet beating [40]. This method proposes steel bullets that can be reused and sterilized, compared to single-use ceramic or glass beads.…”

“…Methods 1 and 3 only differ from a purification method point of view. The purification method appears to make little difference from a DNA extraction point of view, but the commercial kit took 60 min compared to 75 min for the phenol-chloroform purification [40].…”

“…Overall, the superior method time and money-wise was Method 3, incorporating the steel bullet, the lysis buffer, and the commercial kit, making it a suitable option to be further explored [40].…”

“…It is relevant to mention that the authors of the study used an ex vivo model for onychomycosis and that the results can be highly impacted based on preparation [40,55].…”

Development of the Method for Assessing the Viability of Fungi Using the Polymerase Chain Reaction

“…Одним із сучасних та перспективних методів діагностики оніхомікозів вважається молекулярно-генетичний аналіз. Численні дослі дження, проведені з використанням молекулярно-генетичного методу, довели, що пряме виділення ДНК є більш чутливим, точним та швидким, ніж традиційні методи, що базуються на культуральному дослі дженні [8]. Ефективний метод полімераної ланцюгової реакції (ПЛР) розроблений для швидкого виявлення видів дерматофітів безпосередньо з клінічних зразків протягом 24 годин.…”