Mohammadi Parisa, Hamidkhani Aida, Asgarani Ezat
Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, IR Iran.
Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, IR Iran.
Jundishapur J Microbiol. 2015 Oct 12;8(10):e22249. doi: 10.5812/jjm.22249. eCollection 2015 Oct.
Candida species are usually opportunistic organisms that cause acute to chronic infections when conditions in the host are favorable. Accurate identification of Candida species is an essential pre-requisite for improved therapeutic strategy. Identification of Candida species by conventional methods is time-consuming with low sensitivity, yet molecular approaches have provided an alternative way for early diagnosis of invasive candidiasis. Denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient gel electrophoresis (TTGE) are polymerase chain reaction (PCR)-based approaches that are used for studying the community structure of microorganisms. By using these methods, simultaneous identification of multiple yeast species will be possible and reliable results will be obtained quickly.
In this study, DGGE and TTGE methods were set up and evaluated for the detection of different Candida species, and their results were compared.
Five different Candida species were cultured on potato dextrose agar medium for 24 hours. Next, total DNA was extracted by the phenol-chloroform method. Two sets of primers, ITS3-GC/ITS4 and NL1-GC/LS2 were applied to amplify the desired regions. The amplified fragments were then used to analyze DGGE and TTGE profiles.
The results showed that NL1-GC/LS2 primer set could yield species-specific amplicons, which were well distinguished and allowed better species discrimination than that generated by the ITS3-GC/ITS4 primer set, in both DGGE and TTGE profiles. All five Candida species were discriminated by DGGE and TTGE using the NL1-GC/LS2 primer set.
Comparison of DGGE and TTGE profiles obtained from NL1-GC/LS2 amplicons exhibited the same patterns. Although both DGGE and TTGE techniques are capable of detecting Candida species, TTGE is recommended because of easier performance and lower costs.
念珠菌属通常是机会性致病微生物，当宿主条件适宜时可引起急性至慢性感染。准确鉴定念珠菌属是改进治疗策略的必要前提。传统方法鉴定念珠菌属耗时且灵敏度低，而分子方法为侵袭性念珠菌病的早期诊断提供了另一种途径。变性梯度凝胶电泳（DGGE）和温度梯度凝胶电泳（TTGE）是基于聚合酶链反应（PCR）的方法，用于研究微生物群落结构。通过使用这些方法，可以同时鉴定多种酵母菌种，并快速获得可靠结果。
本研究建立并评估了DGGE和TTGE方法用于检测不同念珠菌属菌种，并比较了它们的结果。
将五种不同的念珠菌属菌种在马铃薯葡萄糖琼脂培养基上培养24小时。接下来，采用酚-氯仿法提取总DNA。应用两组引物，即ITS3-GC/ITS4和NL1-GC/LS2来扩增所需区域。然后将扩增片段用于分析DGGE和TTGE图谱。
结果表明，在DGGE和TTGE图谱中，NL1-GC/LS2引物组可产生种特异性扩增子，与ITS3-GC/ITS4引物组相比，这些扩增子区分度更好，能实现更好的菌种鉴别。使用NL1-GC/LS2引物组，通过DGGE和TTGE鉴别出了所有五种念珠菌属菌种。
从NL1-GC/LS2扩增子获得的DGGE和TTGE图谱比较显示出相同的模式。虽然DGGE和TTGE技术都能够检测念珠菌属菌种，但由于操作更简便且成本更低，推荐使用TTGE。
