XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:93-2

Poster (Painel)

93-2

Study of gene amplification in Paracoccidioides brasiliensis by FT-IR

Authors:

Maiara Lima Castilho (UNIVAP - UNIVERSIDADE DO VALE DO PARAIBA) ; Jessica Camila da Silva (UNIVAP - UNIVERSIDADE DO VALE DO PARAIBA) ; Flavia Villaça Morais (UNIVAP - UNIVERSIDADE DO VALE DO PARAIBAUNIP - UNIVERSIDADE PAULISTA) ; Renata de Azevedo Canevari (UNIVAP - UNIVERSIDADE DO VALE DO PARAIBA) ; Maria Angelica Gargione Cardoso (UNIVAP - UNIVERSIDADE DO VALE DO PARAIBA) ; Airton Abrahão Martin (UNIVAP - UNIVERSIDADE DO VALE DO PARAIBA) ; Leandro Raniero (UNIVAP - UNIVERSIDADE DO VALE DO PARAIBA)

Abstract

Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis (PCM). The human infection usually occurs by P. brasiliensis inhaling micelia or yeast contact with damaged tissue. Currently, the mycological diagnosis of PCM is the identification of the typical yeast form of the fungus through directly examination of skin lesions or washed bronchial phlegm, and lung biopsies of patients. However, these routine diagnostic methods often have low sensitivity with a high percentage of false-negatives. Therefore, the objective of this study was to amplify polymorphic genes that are characteristic of P. brasiliensis and study them for FT-IR, Nanodrop®, and agarose gel electrophoresis. DNA extraction was performed according to the phenol-chloroform protocol. The DNA was quantified by optical spectroscopy and displayed on 1% electrophoresis agarose gel, stained with ethidium bromide. The polymerase chain reaction (PCR) for glycoprotein genes, GP43 and GP27 along with ribosomal RNA (rRNA), was conducted in a automatic thermocycler with a final volume of 50 µl containing 50 ng genomic DNA, 200 nmol of each primer and PCR Supermix. The oligonucleotide primers were designed for specifically conserved regions of three genes of P. brasiliensis. The extracted DNA had good performance and integrity. The relationship between the peaks 280nm/260nm had a high purity of DNA and these results were confirmed by electrophoresis. The infrared spectrum obtained from total DNA of the fungus with the amplified DNAs from three genes were compared and correlated with the results of electrophoresis. The results made the identification safer and more accurate for the fungus by amplifying DNA sequences and their comparison by FT-IR, provided through the vibrational modes of biochemical components in the sample, and can, therefore, contribute to the standardization for identifying Pb by FT-IR.਀匀甀瀀漀爀琀攀搀戀礀䘀䄀倀䔀匀倀

Keyword: FT-IR, diagnostic, DNA

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