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Correspondence |

Direct Identification of Dermatophyte DNA From Clinical Specimens by a Nested Polymerase Chain Reaction Assay

Chia-Yi Yang, MD; Tzu-Lung Lin, PhD; Tien-Yi Tzung, MD; Li-Chen Cheng, MD; Jin-Town Wang, MD, PhD; Shiou-Hwa Jee, MD, PhD
Arch Dermatol. 2007;143(6):799-816. doi:10.1001/archderm.143.6.799.
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Fungi have recently emerged as human pathogens capable of causing life-threatening diseases in immunocompromised and other high-risk patients.1 It is therefore advantageous to be able to rapidly and correctly diagnose the fungus.2 Conventional diagnosis is based on phenotypic assignment from fungal cultures. This approach is highly technique dependent, time consuming, and is also largely dependent on the viability and amount of fungal elements in the specimens.3 In this study, we introduce a nested polymerase chain reaction (PCR) assay that can be directly applied to clinical specimens. In addition to the dramatic reduction in diagnosis time, from weeks in fungal culture–based phenotypic identification to 24 to 48 hours, our genotype-based approach affords greatly improved identification accuracy and heightened detection sensitivity.4

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Figure 1.

Representation of the ribosomal DNA (rDNA) gene complex in fungi denoting gene order and the position of the internal transcribed spacer (ITS). Primers were designed on this complex and were designated as ITS1, ITS2, ITS3, and ITS4 as their locations on genes. Oligonucleotide primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS3 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′) were both designed on 18S rDNA. The primer ITS2 (5′-GCT GCG TTC TTC ATC GAT GC-3′) was designed on 5.8S rDNA, and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) was designed on 28S rDNA. Oligonucleotide primer pairs ITS3 and ITS4 were used in the first-round polymerase chain reaction (PCR) amplification reaction. Then the templates of nested PCR were the products of first-round PCR using ITS1 and ITS2 as paired primers.

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Figure 2.

Genomic DNA samples extracted from 5 different skin scrapings were amplified by a MagNA pure Light Cycler System (Roche Diagnostics GmbH, Mannheim, Germany) with nested polymerase chain reaction (PCR) and analyzed by 2% agarose gel electrophoresis. The left panel represents the first round of PCR, while the right panel represents the second round. L and L′ represent the 100–base pair (bp) DNA ladder; 1 and 1′ through 5 and 5′, 5 different skin scraping tests; 6 and 6′, negative controls (Staphylococcus epidermidis genome); and 7 and 7′, positive controls (Trichophyton mentagrophytes genome). All of the distinguishable molecular weights of first PCR products were about 750 bp (lanes 5 and 7); nested PCR products, 350 bp (lanes 1′-5′ and 7′). These 5 clinical specimens belong to the genera Trichophyton and Microsporum and were proved by DNA sequencing. Bands of molecular weight that were less than 200 bp were noted in the negative control and clinical specimens (lane 1′ and lane 6′).

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