Journal of clinical laboratory analysis · 2022
Evaluation of different DNA extraction methods based on steel-bullet beating for molecular diagnosis of onychomycosis.
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Motamedi Marjan, Amini Abdulbaqi, Yazdanpanah Somayeh, Mahmoodi Mozhgan, Khodadadi Hossein, Zalpoor Hamidreza
Аннотация
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.
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.