Molecular identification of Paracoccidioides brasiliensis by 5' nuclease assay Camile P. Semighini a , Zoilo P. de Camargo b , Rosana Puccia b , Maria Helena S. Goldman c , Gustavo H. Goldman a, * a Faculdade de Cie ˆncias Farmace ˆuticas de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, Ribeira ˜o Preto, Sa ˜o Paulo, Brazil b Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa ˜o Paulo (UNIFESP) c Faculdade de Filosofia, Cie ˆncias e Letras de Ribeira ˜o Preto, USP, Sa ˜o Paulo, SP, Brazil Received 3 April 2002; accepted 31 July 2002 Abstract A PCR assay based on the 5' nuclease assay using a fluorescent probe derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis. The assay could detect at least 10 copies of this DNA sequence, providing efficient accuracy to be useful for diagnosis of paracoccidioidomycosis. © 2002 Elsevier Science Inc. All rights reserved. Introduction Paracoccidioides brasiliensis, a thermodimorphic fun- gus, is the causative agent of the most prevalent systemic mycosis in Latin America, paracoccidioidomycosis (PCM). Epidemiologic data show a broad geographic distribution in the Central and South America, from Mexico to Argentina, occurring mainly in Colombia, Venezuela, and Brazil (for a review, see San-Blas and Nin ˜o-Vega, 2001). The main pro- cedures for diagnosis of PCM are: (i) the isolation of the fungus in culture and the positive identification of multibud- ding and birefringent yeast cells by direct examination of biologic fluids or biopsy; and (ii) detection of antibodies. The serologic diagnosis has been extensively used, but some patients present low levels or an absence of detectable antibodies (Del Negro et al., 1991). The main antigenic component in P. brasiliensis is gp43, an exocellular glyco- protein containing a single oligosaccharide chain (Cisalpino et al., 1996; Morais et al 2000). Most sera from patients with PCM recognize this immune-dominant antigen (Blotta and Camargo, 1993). The gene codes for a precursor protein of 416 amino acids, which includes a leader peptide of 35 residues (Cisalpino et al., 1996). Recently, specific molec- ular markers for P. brasiliensis PCR identification using either the gp43 or ribosomal DNA were developed (Bialek et al., 2000; Gomes et al., 2000; Motoyama et al., 2000). PCR has proven to be a powerful tool for quantitative nucleic acid analysis. Several amplification methods, such as quantitative PCR, competitive PCR, and more recently 5' nuclease assays (real-time PCR) have been developed to measure input target templates quantitatively (for a review, see Lie and Petropoulos, 1998). The real-time PCR detec- tion is based on the measurement of fluorescence during the PCR, in which product formation is continuously monitored and validated. The amount of emitted fluorescence is pro- portional to the amount of PCR product and enables the monitoring of the PCR reaction. The real-time PCR method offers several advantages: (i) highly sensitive assay; (ii) it is performed in a closed-tube system and requires no post- PCR manipulation of sample, so increasingly sample throughput; and (iii) it supports the use of a normalization gene for quantitative PCR or house-keeping genes for quan- titative RT-PCR controls (Heid et al., 1996). The 5'-nuclease assay uses Taq-Man self-reporting flu- orescent probes, which are linear oligonucleotides that con- tain a 5' reporter dye and 3' acceptor with overlapping emission-absorption spectra. The reporter dye remains quenched by the 3' acceptor through a Foster-type energy transfer while hybridized to its target. Cleavage of the 5' reporter by 5' nuclease activity of Taq DNA polymerase results in strong fluorescence signals. The development of a specific fluorescent probe for P. brasiliensis would be useful * Corresponding author. Tel.: +1-55-16-6024280; fax: +1-55-16- 6331092 E-mail address: ggoldman@usp.br (G. H. Goldman). Diagnostic Microbiology and Infectious Disease www.elsevier.com/locate/diagmicrobio 44 (2002) 383–386 0732-8893/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0732-8893(02)00472-8