Gnat S, Nowakiewicz A, Ziółkowska G, Trościańczyk A, Majer-Dziedzic B, Zięba P
Sub-Department of Veterinary Microbiology, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland.
State Veterinary Laboratory, Lublin, Poland.
J Appl Microbiol. 2017 May;122(5):1368-1379. doi: 10.1111/jam.13427. Epub 2017 Apr 3.
Recent molecular methods for diagnosis of superficial mycoses have determined the need for a rapid and easy method of extracting DNA. The aim of study was to determine growth conditions and techniques of DNA extraction for Microsporum canis, Trichophyton mentagrophytes and T. verrucosum.
Samples were prepared of each of the DNA extraction methods (phenol-chloroform, CTAB and four different kits) for all of the incubation periods (4, 7 and 10 days) of the cultures on the solid and in the liquid medium. The highest DNA concentrations were obtained using the phenol-chloroform method. The concentration of DNA extracted with the CTAB method accounted for 62·21%, for kits it corresponded from 35·53 to 15·41%. The analysis of the DNA weight yield revealed the highest isolation efficiency of the phenol-chloroform method, 1 mg of mycelium yielded 223·8 μg DNA. Lower DNA yield (by 39·32%) was obtained with the CTAB method; in the case of kits by 68·46-85·32%. In most of the techniques, the DNA yield on the solid medium was higher.
In summary, the highest DNA yield was noted in the 7-day cultures and extraction with the phenol-chloroform method. Importantly, the type of culture was not relevant for the diagnostic result.
Most mycoses are caused by fungi that reside in nature. The severity of the infection depends on the pathogenic attributes, socioeconomic factors and local environmental conditions. Recent diagnosis increasingly relies on not only the clinical features. Molecular identifications have determined the need for a rapid and easy method of extracting DNA. Usually two factors have to be considered: maximize the DNA yield and ensure that the extracted DNA is susceptible to enzymatic reactions. These data suggest that phenol-chloroform methods and a 7-day culture period may be useful for validation and constitute the first step of molecular diagnosis of dermatophytes.
近期用于诊断浅表真菌病的分子方法确定了对一种快速简便的DNA提取方法的需求。本研究的目的是确定犬小孢子菌、须癣毛癣菌和疣状毛癣菌的生长条件及DNA提取技术。
针对在固体和液体培养基上培养的所有培养期（4天、7天和10天），对每种DNA提取方法（苯酚 - 氯仿法、十六烷基三甲基溴化铵法和四种不同试剂盒）都制备了样本。使用苯酚 - 氯仿法获得了最高的DNA浓度。用十六烷基三甲基溴化铵法提取的DNA浓度占62.21%，试剂盒提取的浓度为35.53%至15.41%。DNA产量分析显示苯酚 - 氯仿法的分离效率最高，1毫克菌丝体可产生223.8微克DNA。十六烷基三甲基溴化铵法的DNA产量较低（低39.32%）；试剂盒提取的情况则低68.46% - 85.32%。在大多数技术中，固体培养基上的DNA产量更高。
总之，在7天培养物中采用苯酚 - 氯仿法提取时DNA产量最高。重要的是，培养类型与诊断结果无关。
大多数真菌病由自然界中的真菌引起。感染的严重程度取决于致病属性、社会经济因素和当地环境条件。近期诊断越来越不仅依赖于临床特征。分子鉴定确定了对一种快速简便的DNA提取方法的需求。通常必须考虑两个因素：最大化DNA产量并确保提取的DNA易于进行酶促反应。这些数据表明苯酚 - 氯仿法和7天培养期可能有助于验证，并且是皮肤癣菌分子诊断的第一步。
