말라쎄지아 효모균의 분자생물학적 분석으로서집락 중합효소연쇄반응의 적용
The Application of Colony PCR in the Molecular Biological Analysisof Malassezia Yeasts
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Background: Malassezia yeasts are lipophilic fungi that are found in 75~80% of healthy adults. The yeasts are known to be associated with pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and recently its pathogenicity is being expanded to other various skin disorders, such as atopic dermatitis and acne vulgaris. Recently, various molecular biological techniques are being preferred over morphological analysis. In order to perform a DNA-based diagnostic test, availability of a simple, rapid, and reliable DNA extraction protocol is essential. Objective: We sought to implement novel molecular biology technique, namely colony PCR method using microwave as the easiest way to amplification of Malassezia target DNA, and assess its clinical applicability. Methods: Instead of using templates of purified genomic DNA, we performed the PCR directly from Malassezia colonies. A fresh yeast colony transferred to the bottom of a microcentrifuge tube and microwaved for 1 min three times in the presence of a pyrex beaker containing 50 ml of sterile water to dissipate excess heat. Following this microwave lysis, PCR-reaction mixture was added directly to the microcentrifuge tube. Two DNA extraction methods (boiling method, glass beads method) were used for comparing the sensitivity and effectiveness with the colony PCR method. All reactions were performed using the primers 26S and ITS1 complementary to the rDNA region. Results 1. As a result of gel electrophoresis, we recognized expected PCR products (approximately 580 bp for 26S rDNA and 250~320 bp for ITS1) from both colony PCR method and two DNA extraction methods (boiling method, glass beads method). 2. As a result of measuring nucleic acid level with the spectrophotometer, colony PCR disregarding DNA extraction process shows relatively similar PCR efficacy compared with the boiling and glass beads method. And there is no significant difference among those methods statistically (p>0.001). 3. In conducting the PCR method, boiling method required approximately 400 minutes, and glass beads method required approximately 360 minutes, respectively. As contrasted with two methods, colony PCR method required approximately 150 minutes, and could be capable of saving time. In addithion, colony PCR had an economic efficiency comparing with boiling method and glass beads methods. Conclusions: All these findings suggest that directly application of the Malassezia yeasts obtained from culture colony for PCR reaction is a fast, reliable, cost-effective and simple method for performing any PCR-based protocol including diagnostic tests.
Abstract
Background: Malassezia yeasts are lipophilic fungi that are found in 75~80% of healthy adults. The yeasts are known to be associated with pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and recently its pathogenicity is being expanded to other various skin disorders, such as atopic dermatitis and acne vulgaris. Recently, various molecular biological techniques are being preferred over morphological analysis. In order to perform a DNA-based diagnostic test, availability of a simple, rapid, and reliable DNA extraction protocol is essential. Objective: We sought to implement novel molecular biology technique, namely colony PCR method using microwave as the easiest way to amplification of Malassezia target DNA, and assess its clinical applicability. Methods: Instead of using templates of purified genomic DNA, we performed the PCR directly from Malassezia colonies. A fresh yeast colony transferred to the bottom of a microcentrifuge tube and microwaved for 1 min three times in the presence of a pyrex beaker containing 50 ml of sterile water to dissipate excess heat. Following this microwave lysis, PCR-reaction mixture was added directly to the microcentrifuge tube. Two DNA extraction methods (boiling method, glass beads method) were used for comparing the sensitivity and effectiveness with the colony PCR method. All reactions were performed using the primers 26S and ITS1 complementary to the rDNA region. Results 1. As a result of gel electrophoresis, we recognized expected PCR products (approximately 580 bp for 26S rDNA and 250~320 bp for ITS1) from both colony PCR method and two DNA extraction methods (boiling method, glass beads method). 2. As a result of measuring nucleic acid level with the spectrophotometer, colony PCR disregarding DNA extraction process shows relatively similar PCR efficacy compared with the boiling and glass beads method. And there is no significant difference among those methods statistically (p>0.001). 3. In conducting the PCR method, boiling method required approximately 400 minutes, and glass beads method required approximately 360 minutes, respectively. As contrasted with two methods, colony PCR method required approximately 150 minutes, and could be capable of saving time. In addithion, colony PCR had an economic efficiency comparing with boiling method and glass beads methods. Conclusions: All these findings suggest that directly application of the Malassezia yeasts obtained from culture colony for PCR reaction is a fast, reliable, cost-effective and simple method for performing any PCR-based protocol including diagnostic tests.
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