title: Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens.
abstract: An accurate diagnosis of tinea unguium is necessary for the selection of antimycotics and successful treatment. To rapidly and accurately identify the aetiological agents causing tinea unguium, we improved upon the conventional boiling method for DNA extraction and developed a novel real-time PCR detection system that includes two assays. The two assays, based on the amplification of ribosomal internal transcribed spacer regions and 28S rDNA, were designed to detect pan-dermatophyte and Trichophyton rubrum, respectively. The analytical sensitivities of both assays permitted the detection of ten copies of plasmid DNA template. The analytical specificity of the detection system was confirmed using 11 dermatophyte strains and 25 non-dermatophyte strains. In total, 165 nail specimens were examined by microscopy, culture, conventional PCR, and the novel real-time PCR method. Real-time PCR gave positive results in 47.3 % of the specimens (78), a rate exceeding those obtained using microscopy (72, 43.6 %), conventional PCR (69, 41.8 %), and culture (49, 29.7 %). All conventional PCR-positive specimens were detected by real-time PCR, and real-time PCR detected nine specimens that were missed by conventional PCR. The results from latent class analysis, and further calculations, showed that real-time PCR was the most sensitive method, but the diagnostic specificity of the four approaches was equivalent. In particular, molecular approaches may be more effective than microscopy and culture when the clinical symptoms of tinea unguium are not evident.
authors.affiliation: Department of Dermatology, Peking University First Hospital, No. 8 Xishiku St., West District, Beijing, 100034, People's Republic of China.|Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, 100034, People's Republic of China.|Research Center for Medical Mycology, Peking University, Beijing, 100034, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Gong
authors.affiliation: Department of Dermatology, Peking University First Hospital, No. 8 Xishiku St., West District, Beijing, 100034, People's Republic of China.
authors.forename: Menglong
authors.initials: M
authors.lastname: Ran
authors.affiliation: Department of Dermatology, Peking University First Hospital, No. 8 Xishiku St., West District, Beijing, 100034, People's Republic of China.|Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, 100034, People's Republic of China.|Research Center for Medical Mycology, Peking University, Beijing, 100034, People's Republic of China.
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Dermatology, Peking University First Hospital, No. 8 Xishiku St., West District, Beijing, 100034, People's Republic of China.|Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, 100034, People's Republic of China.|Research Center for Medical Mycology, Peking University, Beijing, 100034, People's Republic of China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Wan
authors.affiliation: Department of Dermatology, Peking University First Hospital, No. 8 Xishiku St., West District, Beijing, 100034, People's Republic of China. lrymm@medmail.com.cn.|Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, 100034, People's Republic of China. lrymm@medmail.com.cn.|Research Center for Medical Mycology, Peking University, Beijing, 100034, People's Republic of China. lrymm@medmail.com.cn.
authors.forename: Ruoyu
authors.initials: R
authors.lastname: Li
mesh_terms: D003883:Arthrodermataceae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D002681:China; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification; D021903:DNA, Ribosomal Spacer / Q000235:genetics / Q000302:isolation & purification; D006785:Hospitals, University; D006801:Humans; D008828:Microbiological Techniques / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009262:Nails / Q000382:microbiology*; D014009:Onychomycosis / Q000175:diagnosis* / Q000382:microbiology*; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods
chemical_list: D004271:DNA, Fungal; D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer; D012339:RNA, Ribosomal, 28S
keywords: Dermatophyte PCR; Fungal infection; Pan-dermatophyte; Tinea unguium