Kenjar Apoorva, M Raj Juliet R, Bhandary Joshika, Girisha Banavasi S, Chakraborty Gunimala, Karunasagar Indrani
Division of Infectious Diseases, Nitte University Center for Science Education and Research, Mangaluru, Karnataka, India.
Department of Dermatology, K S Hegde Medical Academy, Mangaluru, Karnataka, India.
Indian J Dermatol. 2021 Nov-Dec;66(6):668-673. doi: 10.4103/ijd.ijd_19_21.
Polymerase chain reaction (PCR) is the most optimized method for the rapid detection and analysis of any environmental or clinically significant organism. While PCR amplification directly from samples has been shown effective for several bacteria and viruses, for filamentous fungus and yeast, extraction of genomic DNA is a must. The extraction of DNA from fungal cultures is often reported using user-friendly commercially available kits, which are designed to decrease the time, extensive manual work in extraction procedures but are often expensive. Dermatophytes pose an added drawback to efficient DNA extraction due to their poor recovery on culture media and slow growth rate.
In the present study, we developed and validated a method for effective genomic DNA extraction from dermatophytes.
DNA yield from standard dermatophytes extracted from spore suspensions and mycelia mat by commercially available kits was compared. A modified method using lyticase buffer and phenol-chloroform extraction was developed. The yield obtained was compared with the existing methods (kit-based method and cetyl trimethyl ammonium bromide method). The yield and quality of the total genomic DNA were estimated spectrophotometrically and by successful PCR amplification of the ITS region. The results were validated using 21 clinical isolates from recalcitrant dermatophytosis.
Minimal fungal DNA was obtained from the spores compared to that obtained from mycelial mat. Commercially available kits yielded lower amounts of DNA compared to the CATB method. The modified method developed in this study yielded better quality and quantity of DNA.
Of the three extraction methods evaluated, the developed method gave significantly higher total genomic DNA yield and better purity than the reference methods. In addition, the turnaround time for DNA extraction was reduced to half based on modifications in culture conditions.
聚合酶链反应（PCR）是用于快速检测和分析任何具有环境或临床意义的生物体的最优化方法。虽然直接从样本进行PCR扩增已被证明对多种细菌和病毒有效，但对于丝状真菌和酵母而言，基因组DNA的提取是必不可少的。从真菌培养物中提取DNA的方法通常报道为使用便于用户操作的市售试剂盒，这些试剂盒旨在减少提取过程中的时间和大量手工操作，但往往价格昂贵。皮肤癣菌由于在培养基上回收率低且生长速度缓慢，给高效DNA提取带来了额外的困难。
在本研究中，我们开发并验证了一种从皮肤癣菌中有效提取基因组DNA的方法。
比较了使用市售试剂盒从孢子悬液和菌丝体垫中提取的标准皮肤癣菌的DNA产量。开发了一种使用溶菌酶缓冲液和苯酚 - 氯仿提取的改良方法。将获得的产量与现有方法（基于试剂盒的方法和十六烷基三甲基溴化铵方法）进行比较。通过分光光度法以及对ITS区域进行成功的PCR扩增来估计总基因组DNA的产量和质量。使用来自顽固性皮肤癣菌病的21株临床分离株对结果进行验证。
与从菌丝体垫获得的DNA相比，从孢子中获得的真菌DNA极少。与CATB方法相比，市售试剂盒产生的DNA量较低。本研究开发的改良方法产生了质量和数量更好的DNA。
在评估的三种提取方法中，所开发的方法比参考方法产生的总基因组DNA产量显著更高且纯度更高。此外，基于培养条件的改进，DNA提取的周转时间减少到了一半。
