The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high-resolution melting curve analysis
The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high-resolution melting curve analysis
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DOI：
10.1111/omi.12050
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发表时间：
2014-06-01
影响因子：
3.7
通讯作者：
McCullough, M. J.
中科院分区：
文献类型：
--
作者：
Alnuaimi, A. D.;Wiesenfeld, D.;McCullough, M. J.
准确、快速、经济的真菌种鉴定一直是真菌学研究的主要目标。在这项研究中，我们的目标是检查的核糖体DNA（rDNA）的内部转录区ITS1和ITS2的高分辨率熔解曲线分析（HRMA）的可行性，快速，简单和廉价的8个临床相关念珠菌（白色念珠菌，光滑念珠菌，近平滑念珠菌，克鲁斯念珠菌，热带念珠菌，季也蒙念珠菌，都柏林念珠菌和葡萄牙念珠菌）的分化。此外，我们第一次测试了HRMA的适用性，使用跨越rDNA的25 S的转座内含子区域的引物组将白色念珠菌菌株分为四种先前描述的基因型（A、B、C和D）。本研究使用了典型和未知的临床口腔分离株，并将熔解曲线分析与扩增子测序和琼脂糖凝胶电泳分析进行了比较。两个rDNA区域的实时PCR和随后的HRMA产生了不同的熔解曲线，其与测序和凝胶电泳分析雅阁，具有高度的可重复性，并且是8种念珠菌属和白色念珠菌基因型中每一种的特征。此外，在4h内获得结果，并且不需要任何扩增后处理，因此减少了时间和成本。由于其简单和快速，该技术是一个很好的适合在大流行病学背景下的数百个临床菌株的基因型分析。
Accurate, rapid and economical fungal species identification has been a major aim in mycology. In this study, our goal was to examine the feasibility of a high-resolution melting curve analysis (HRMA) of internal transcribed regions ITS1 and ITS2 in ribosomal DNA (rDNA) for a rapid, simple and inexpensive differentiation of eight clinically relevant Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Candida guilliermondii, Candida dubliniensis and Candida lusitaniae). In addition, for the first time, we tested the applicability of HRMA to classify C.albicans strains into four previously described genotypes (A, B, C and D) using a primer set that spans the transposable intron region of 25S of rDNA. Type and unknown clinical oral isolates were used in this study and the melting curve analysis was compared with both amplicons' sequencing and agarose gel electrophoresis analysis. Real-time PCR and subsequent HRMA of the two described rDNA regions generated distinct melting curve profiles that were in accord with sequencing and gel electrophoresis analysis, highly reproducible, and characteristic of each of the eight Candida species and C.albicans genotypes. Moreover, results were obtained in 4h and without the need for any post-amplification handling, so reducing time and cost. Owing to its simplicity and speed, this technique is a good fit for genotypic analysis of hundreds of clinical strains in large epidemiological settings.