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

Abstract

Introduction: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis. Methods: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis. Results: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis. Conclusions: The new semi-nested PCR describe is useful for paracoccidioidomycosis diagnosis.

An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection

“…The limit of detection for the OTsn-PCR assay was comparable with that of the protocol developed by Koishi et al 5 ( Figure 1B) and was more sensitive than the protocols in the Imai et al 8 and Theodoro et al 9 studies (Figures 1C-1D). Furthermore, when using DNA extracted from the clinical samples, the OTsn-PCR assay and the protocol developed by Koishi et al 5 were able to detect P. brasiliensis DNA in all of the samples (100%) ( Table 1), while the protocols of Imai et al 8 and Theodoro et al 9 only detected P. brasiliensis DNA in four (28.5%) and eight (57.2%) of the clinical samples, respectively (Table 1). A possible cause for this difference may be the lower numbers of cycles used in these reactions.…”

“…However, in our study, we achieved the same limit of detection for the OTsn-PCR assay as the semi-nested PCR approach described by Koishi et al 5 , which was only possible due to the use of gelatin in the reaction. When the OTsn-PCR assay was performed without gelatin, the limit of DNA detection was 0.25ng (data not shown).…”

“…For this study, we used the following microorganisms: P. brasiliensis (LDR 1 and Pb 18 strains), Cryptococcus sp., Sporothrix spp., Histoplasma capsulatum, Candida albicans Deoxyribonucleic acid ( DNA) from yeast-phase fungal cells and clinical samples were extracted by cell lysis via a liquid nitrogen freeze-thaw, followed by phenol-chloroform treatment and sodium acetate-ethanol precipitation as described by Koishi et al 5 The DNA concentration and purity were determined by spectrophotometry measurement at 260/280nm. Due to the possible presence of inhibitors, the quality of DNA extracted from the clinical samples was evaluated by PCR using two primers specifi c to the human CXCR4 gene; SDF1 (5'-CAGTCAACCTGGGCAAAGCC-3') and SDF2 (5'-CCTGAGAGTCCTTTTGCGGG-3') 6 .…”

“…This OTsn-PCR approach was compared with the nested and semi-nested PCR methods described by Imai et al 8 , Theodoro et al 9 and Koishi et al 5 Similar to our OTsn-PCR assay, these reactions use sequences of ribosomal DNA as targets, especially from the ITS, and are specifi c for the detection of P. brasiliensis. For the sake of comparison, we used fi ve different serially diluted P. brasiliensis DNA concentrations (2.5ng, 250pg, 25pg, 2.5pg and 0.25pg).…”

Lung Diseases - Selected State of the Art Reviews

“…These laboratory tests have great value and can also be employed in follow-up studies (Gómez et al, 1997Lacaz et al, 2002;Nucci et al, 2009;Restrepo et al, 2009Restrepo et al, , 2011. In addition, in the last decade, molecular tests to diagnose PCM have been implemented; these tests require further research (Buitrago et al, 2011;Gomes et al, 2000;Bialek et al, 2000a;Koishi et al, 2010). …”