Reverse Transcription-3′ Rapid Amplification of cDNA Ends-nested PCR of ACT1 and SAP2 mRNA as a Means of Detecting Viable Candida albicans in an In Vitro Cutaneous Candidiasis Model1  Charles N. Okeke, Ryoji Tsuboi, Masaaki Kawai, Masashi Yamazaki, Suttirat Reangchainam, Hideoki Ogawa  Journal of Investigative Dermatology  Volume 114, Issue 1, Pages 95-100 (January 2000) DOI: 10.1046/j.1523-1747.2000.00845.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 A schematic representation of the reverse transcription-3′ RACE-nested PCR protocol. The 3′ RACE anti-sense primer, M13 M4′, is complementary with the adaptor primer, M13 M4, ligated to the poly (dT) primer; P1, represents the 3′ RACE anchor sense primer; P2 and P3 represent the nested PCR sense and anti-sense primers. Journal of Investigative Dermatology 2000 114, 95-100DOI: (10.1046/j.1523-1747.2000.00845.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Detection of amplification products of ACT1 and SAP2. The mRNA and DNA were extracted from C. albicans (JUH 3181) and amplified by reverse transcription-3′ RACE-nested PCR and PCR, respectively, as described in the text. Following electrophoresis through agarose gel, the amplified products were visualized by staining with ethidium bromide. Lane M, size marker (ΦX 174/HinfI digest); lane 1, ACT1 (3′ RACE); lane 2, ACT1 (nested PCR); lane 3, ACT1 (DNA PCR); lane 4, SAP2 (3′ RACE); lane 5, SAP2 (nested PCR); lane 6, SAP2 (DNA PCR). The expected sizes of the ACT1 and SAP2 amplicons are 315 and 162 bp, respectively. The locations of some of the DNA standards (bp) are indicated (lane M). Journal of Investigative Dermatology 2000 114, 95-100DOI: (10.1046/j.1523-1747.2000.00845.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Detection of amplification products of ACT1 and SAP2 from C. albicans cultured in Sabouraud or albumin-supplemented yeast carbon base broths. The mRNA and DNA templates, extracted from C. albicans cultured for 24 h in SB (lanes 1–4) and YCB-BSA (lanes 5–8), were, respectively, subjected to reverse transcription-3′ RACE-nested PCR and PCR amplifications. Lane M, size marker (ΦX 174/HinfI digest); lane 1, ACT1 (PCR); lane 2, ACT1 (nested PCR); lane 3, SAP2 (PCR); lane 4, SAP2 (nested PCR); lane 5, ACT1 (PCR); lane 6, ACT1 (nested PCR); lane 7, SAP2 (PCR); lane 8, SAP2 (nested PCR). Journal of Investigative Dermatology 2000 114, 95-100DOI: (10.1046/j.1523-1747.2000.00845.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Specificity of ACT1 and SAP2 primer pairs by reverse transcription-3′ RACE-nested PCR amplification. Total RNAs extracted from a number of fungal and bacterial species were used as templates for reverse transcription-3′ RACE-nested PCR amplification using the ACT1 or SAP2 primer pairs. The final amplification products were separated by electrophoresis on 1.5% (wt/vol) agarose gel and visualized by ethidium bromide staining. (A), amplification by ACT1 primers; (B), SAP2 primers. Lane M, size marker (ΦX 174/HinfI digest); lane 1, C. albicans;lane 2, C. guilliermondii;lane 3, C. krusei;lane 4, C. parapsilosis;lane 5, C. tropicalis;lane 6, A. flavus;lane 7, A. fumigatus;lane 8, A, niger;lane 9, T. mentagrophytes;lane 10, T. rubrum;lane 11, S. epidermidis;lane 12, Str. sanguis;lane 13, LSE (skin tissue). Journal of Investigative Dermatology 2000 114, 95-100DOI: (10.1046/j.1523-1747.2000.00845.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Sensitivity of SAP2 mRNA detection by reverse transcription-3′ RACE-nested PCR amplification. Reverse transcription-3′ RACE-nested PCR amplification was performed with different quantities of total RNA as templates. (A) The amplified products were electrophoresed on 1.5% (wt/vol) agarose gel and visualized by staining with ethidium bromide. (B) The southern-blotted amplicons from the samples shown in (A) were hybridized with a specific probe labeled with fluorescein-11-dUTP, as described in the text. Lane M, size marker (ΦX 174/HinfI digest); lane 1, 5 ng; lane 2, 1 pg; lane 3, 500 fg; lane 4, 200 fg; lane 5, 100 fg; lane 6, 10 fg; lane 7, 1 fg. Journal of Investigative Dermatology 2000 114, 95-100DOI: (10.1046/j.1523-1747.2000.00845.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Histologic observations of vertical sections of LSE after 5 d of colonization with C. albicans. Sections were stained by Grocott methenamine silver staining method. Scale bar: 0.2 mm. (A) Noninfected LSE; (B) C. albicans-infected and untreated LSE; (C) C. albicans-infected and amorolfine-treated LSE. Journal of Investigative Dermatology 2000 114, 95-100DOI: (10.1046/j.1523-1747.2000.00845.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Detection of amplification products of ACT1 and SAP2 from LSE samples. Total RNA and genomic DNA were extracted from proteinase K-digested samples of LSE shown in Figure 6, as described in the text, and subjected to reverse transcription-3′ RACE-nested PCR and PCR amplification, respectively. Ten microliters of amplified products were separated on 1.5% (wt/vol) agarose gel incorporating ethidium bromide. (A) Noninfected LSE; (B), C. albicans-infected and untreated LSE; (C) 10 μg of amorolfine was applied to the surface of LSE at 48 h postinoculation and into the LSE assay medium at 48 and 96 h postinoculation. Lane M, size marker (ΦX 174/Hinf I digest); lane 1, ACT1 (PCR); lane 2, ACT1 (nested PCR); lane 3, SAP2 (PCR); lane 4, SAP2 (nested PCR). Journal of Investigative Dermatology 2000 114, 95-100DOI: (10.1046/j.1523-1747.2000.00845.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions