Primers for Clinical Detection of Paracoccidioides brasiliensis

Abstract

From a 0.72-kb fragment universally generated in Paracoccidioides brasiliensis strains, primers were designed and tested on genomic DNA of this and other pathogenic fungi. They were specific and highly sensitive for P. brasiliensis DNA. Positive results were obtained when these were tested in clinical samples. Paracoccidioidomycosis (PCM), caused byParacoccidioides brasiliensis, is a chronic granulomatous systemic mycosis prevalent in rural areas of Latin America (16).

A semi-nested PCR assay for molecular detection of Paracoccidioides brasiliensis in tissue samples

“…In a preview study using the ITS1-5.8S-ITS2 region as target, an unexpected cross-reaction with H. capsulatum was observed 6 . In this study, the snPCR was specific for P. brasiliensis and no amplification product was observed with DNA from the other tested fungi; moreover, it showed greater sensitivity than some previous reports 10 and similar to a nested PCR assay for S. schenckii 13 . The high sensitivity in the present test is probably the result of choosing the high copy number ITS1-5.8S-ITS2 region 14 in association with snPCR, recognized as having a 1,000-fold greater sensitivity than conventional PCR.…”

“…PCR assays have been used experimentally to detect P. brasiliensis in the serum and tissues of infected mice 2,4 , in artificially contaminated soil and in environmental samples 8 . In PCM patients, PCR has been used on sputa, cerebrospinal fluid and paraffin-embedded tissues 3,9,10 . However, molecular diagnosis of PCM is not used in clinical routine.…”

Important aspects of oral paracoccidioidomycosis–a literature review

“…71 Some authors report the use of the arbitrary primer OPG18 (Operon Biotechnology, Huntsville, AL, USA), which generates two specific DNA fragments (0.72 and 0.83 kb) common to and specific for all P. brasiliensis. 70 Probes identifying both Blastomyces dermatitidis and P. brasiliensis (AccuProbe, Gen-Probe) are commercially available, with the limitation of requiring microscopic examination to differentiate one fungus from the other. More recently, Buitrago et al [72] developed a molecular diagnostic technique based on real-time PCR for the detection of P. brasiliensis DNA in both culture and patientsÕ clinical samples.…”

Paracoccidioides brasiliensis: chemical and molecular tools for research on cell walls, antifungals, diagnosis, taxonomy

“…One pair from the 0.72 bp fragment MG2(1)F: 5 0 -GGGATTCCCTAGGCAAACA CTTGTGTGA -3 0 ; MG2(1)R: 5 0 -GTGCAGTTATCCACAAGCCA TATATTC -3 0 was specific for P. brasiliensis, and detected as little as 10 pg of fungal DNA [74]. These primers produced positive identification bands in patients with a confirmed diagnosis of chronic PCM (Fig.…”