Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis.
BACKGROUND
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions.


OBJECTIVE
The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus.


METHODS
To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit.


RESULTS
The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal.


CONCLUSIONS
The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological samples in order to validate this method and then generate a diagnostic kit for Paracoccidioidomycosis.
Nucleo de Pos-Graduacao e Pesquisa da Santa Casa de Belo Horizonte, Minas Gerais. Brazil.
Fabiana
F
Rocha-Silva
Centro de Pesquisas Rene Rachou-FIOCRUZ, Belo Horizonte. Brazil.
Luciana I
LI
Gomes
Nucleo de Pos-Graduacao e Pesquisa da Santa Casa de Belo Horizonte, Minas Gerais. Brazil.
Cidiane
C
Gracielle-Melo
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Minas Gerais. Brazil.
Alfredo M
AM
Goes
Rua Domingos Vieira, 590, CEP: 30150240 - Belo Horizonte, Minas Gerais. Brazil.
Rachel B
RB
Caligiorne
D001938:Brazil; D003198:Computer Simulation; D005656:Fungal Proteins / Q000235:genetics*; D016681:Genome, Fungal; D006801:Humans; D010228:Paracoccidioides / Q000235:genetics / Q000302:isolation & purification*; D010229:Paracoccidioidomycosis / Q000175:diagnosis* / Q000382:microbiology; D010330:Patents as Topic; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D005656:Fungal Proteins; C000606073:Pb27 protein, Paracoccidioides brasiliensis
Diagnosis; Paracocciodioides brasiliensis; Paracocciodioides lutzii; Pb 27 protein; paracoccidioidomycosis; real time-PCR.