Application of polymerase chain reaction (PCR) and PCR based restriction fragment length polymorphism for detection and identification of dermatophytes from dermatological specimens

Abstract

Objective: To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes.

Materials and methods: Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods.

Results: PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%.

Conclusion: PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes.

Keywords: 18S rDNA; PCR; PCR-RFLP; dermatophytes.

Fig. 1

(A) Agarose gel electrophoresis showing the application of dermatophyte-specific PCR on dermatological specimens: Lane 1: Negative control, Lane 2: Nail clipping - Positive, Lane 3: Nail clipping - Positive, Lane 4: Nail clipping - Positive, Lane 5: Blister top - Positive, Lane 6: Blister top - Positive, Lane 7: Nail clipping- Positive, Lane 8: Nail clipping - Positive, Lane 9: Nail clipping - Positive, Lane 10: Nail clipping - Positive, Lane 11: Nail clipping - Positive, Lane 12: Nail clipping - Positive, Lane 13: Nail clipping - Positive, Lane 14: Positive control: standard strain of T. rubrum, Lane 15: Molecular weight marker Hinf – I digest of φX174 bacteriophage DNA; (B) Agarose gel electrophoresis showing the application of ITS PCR on dermatological specimens: Lane 1: Negative control, Lane 2: Nail clipping - Positive, Lane 3: Nail clipping - Positive, Lane 4: Nail clipping - Positive, Lane 5: Blister top - Positive, Lane 6: Blister top - Positive, Lane 7: Nail clipping - Positive, Lane 8: Nail clipping - Positive, Lane 9: Nail clipping - Positive, Lane 10: Nail clipping - Positive, Lane 11: Nail clipping - Positive, Lane 12: Nail clipping - Negative, Lane 13: Nail clipping - Positive, Lane 14: Nail clipping - positive, Lane 15: Blister top - Positive, Lane 16: Nail clipping - Positive, Lane 17: Positive control: T. rubrum standard strain DNA, Lane 18: Molecular weight marker – Hae - III φX174 bacteriophage DNA

Fig. 2

Agarose gel electrophoresis of PCR-RFLP performed on dermatophyte-specific PCR amplicons: Lane 1: Digested product of M. gypseum (120, 40 bp), Lane 2: Digested product of T. rubrum VRF 1478/06 (90, 50, 40 bp), Lane 3: Digested product of T. rubrum VRF 1593 /06 (90, 50, 40 bp), Lane 4: Digested product of T. rubrum standard strain, Lane 5: Molecular weight marker Hinf–I digest of φX174 bacteriophage

Fig. 3

(A) Agarose gel electrophoresis of PCR-RFLP performed on ITS PCR amplicons to identify dermatophytes: Lane 1: Undigested product (595 bp), Lane 2: Digested product of M. gypseum (420, 95 bp), Lanes 3, 6, 7, 8 Digested product of T. rubrum (300, 200, 95 bp), Lane 4: Digested product of E. floccosum (350, 95 bp), Lane 9: Digested product of standard strain of T. rubrum, Lane 10: Molecular weight marker Hinf –I digest of φX174 bacteriophage DNA; (b) Agarose gel electrophoresis of PCR-RFLP performed on ITS PCR amplicons to identify nondermatophyte etiology: Lane 1: Undigested product C. albicans ATCC 24433 (595 bp), Lane 2: Digested product identified as A. niger (440, 150 bp), Lane 3: Digested product identified as C. albicans (331, 150, 100 bp), Lane 4: Digested product identified as C. tropicalis (351, 150, 100, 50 bp), Lane 5: Digested product identified as C. parapsilosis (331,150,100,50 bp), Lane 6: Digested product identified as A. fumigatus (301, 160,150 bp), Lane 7: Digested product identified as A. flavus (400, 195 bp), Lane 8: Digested product identified as A. fumigatus (301, 160, 150 bp), mw: Molecular weight marker Hinf –I digest of φX174 bacteriophage DNA