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. 2023 Aug 31;36(2):78-85.
doi: 10.54589/aol.36/2/78.

gDNA extraction from Candida albicans and Candida dubliniensis in subgingival samples in Argentina. Evaluation of different methods

Verónica A Dubois  1   2 , Pablo A Salgado  1   2   3 , Laura A Gliosca  4   2 , Susana L Molgatini  1   2
Affiliations

gDNA extraction from Candida albicans and Candida dubliniensis in subgingival samples in Argentina. Evaluation of different methods

Verónica A Dubois et al. Acta Odontol Latinoam. .

Abstract
in English, Spanish

The oral cavity constitutes a unique ecosystem with highly variable ecological niches that harbor a great variety of microorganisms, including yeasts. Molecular methods are currently considered the gold standard for identifying species, although they involve limitations associated with the disruption of yeast cell walls to release the genomic DNA (gDNA) for amplification.

Aim: The aim of this study was to compare the performance of different methods for extracting gDNA from Candida albicans and Candida dubliniensis, subsequently amplifying DNA by PCR.

Materials and method: Fifty-two isolates (16 C. albicans and 36 C. dubliniensis) were obtained from subgingival biofilm of HIV+ patients with clinical signs of periodontal disease. The study evaluated 6 gDNA extraction methods and two PCR amplification methods. Furthermore, the presence of alleles of HWP1 gene was determined in C. albicans.

Results: Comparisons of six methods show statistically significant differences (p <0.001) except for C. albicans in two of them. For C. dubliniensis, statistical differences were observed in all comparisons. Commercial methods were more efficient for concentrating gDNA than in-house methods, and both PCRs were effective. Ten heterozygous C. albicans isolates for this allele were positive for the HWP1-1 / HWP1-2 allele, one was homozygous for Wild Type HWP1-1 allele, and 5 were homozygous for novel/rare HWP1-2 allele.

Conclusions: This study aims to provide simple, inexpensive strategies for phenotypic identification and molecular confirmation of Candida albicans and Candida dubliniensis for non-reference laboratories with low complexity and/or low budgets.

La cavidad oral constituye un ecosistema único con nichos ecológicos muy variables, capaz de albergar una gran variedad de microorganismos, incluidas las levaduras. Los métodos moleculares son considerados actualmente los métodos de identificación definitivos ya que a diferencia de los anteriores, nos brindan una correcta sensibilidad y especificidad. Sin embargo, existen limitaciones asociadas con la ruptura de las paredes celulares de estas levaduras para liberar el ADN genómico (gADN) necesario para la amplificación.

Objetivo: El objetivo de este estudio fue comparar el rendimiento de diferentes métodos de extracción de gADN de Candida albicans y Candida dubliniensis, amplificando posteriormente por PCR. Materiales y Método: Se estudiaron 52 aislamientos, 16/52 de Candida albicans y 36/52 de Candida dubliniensis obtenidos de biofilm subgingival de pacientes VIH+ con signos clínicos de enfermedad periodontal. Se evaluaron seis métodos de extracción de gADN y la posterior amplificación se realizó por dos técnicas de PCR. Además en C. albicans se determinó la presencia de alelos para el gen HWP1.

Resultados: Las comparaciones de seis métodos son estadísticamente significativas (p<0,001) excepto para C. albicans en dos de ellos. Para C. dubliniensis se observaron diferencias estadísticas en todas las comparaciones. Los métodos comerciales mostraron una mayor eficiencia en la concentración de gADN que los métodos caseros y ambos fueron efectivos en las dos PCR. 10 aislados de C. albicans resultaron positivos para el alelo HWP1-1/HWP1-2, siendo heterocigotos para este alelo. Solo un aislamiento fue homocigoto para el alelo HWP1-1 de tipo salvaje y 5 eran homocigotos para el alelo HWP1-2 nuevo/raro.

Conclusiones: Este estudio tiene como objetivo proporcionar estrategias simples y económicas para la identificación fenotípica y confirmación molecular de Candida albicans y Candida dubliniensis para laboratorios de no referencia con baja complejidad y/o bajo presupuesto económico.

Keywords: Candida albicans; Candida dubliniensis; PCR; gDNA; qPCR; subgingival samples.

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Conflict of interest statement

The authors declare no potential conflicts of interest regarding the research, authorship, and/or publication of this article.

Figures

Fig. 1
Fig. 1. Melting point of C. albicans86°C (+0.5) and C. dubliniens 82° C (+0.5) using as extraction method heating at 100°C in qPCR.
Fig. 2
Fig. 2. 1.3% agarose gel in TAE buffer (Tris, Acetic Acid, EDTA). Block 8: Ladder 100 pb. Lane 1 positive strain for Candida albicans, lane 2 positive strain for Candida dubliniensis lanes 3, 4, 5, 6 negative strains, lane 7 negative control.
See this image and copyright information in PMC

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