RESUMO
ABSTRACT 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.
RESUMEN 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.
RESUMO
Sixteen genomic DNA simple sequence repeat (SSR) markers of Lentinula edodes were developed from 205 SSR motifs present in 46.1-Mb long L. edodes genome sequences. The number of alleles ranged from 3–14 and the major allele frequency was distributed from 0.17–0.96. The values of observed and expected heterozygosity ranged from 0.00–0.76 and 0.07–0.90, respectively. The polymorphic information content value ranged from 0.07–0.89. A dendrogram, based on 16 SSR markers clustered by the paired hierarchical clustering' method, showed that 33 shiitake cultivars could be divided into three major groups and successfully identified. These SSR markers will contribute to the efficient breeding of this species by providing diversity in shiitake varieties. Furthermore, the genomic information covered by the markers can provide a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection in the shiitake mushroom.
Assuntos
Alelos , Cruzamento , DNA , Frequência do Gene , Ligação Genética , Variação Genética , Genoma , Lentinula , Métodos , Repetições de Microssatélites , Cogumelos ShiitakeRESUMO
The identification of traditional Chinese herbs(TCH) is very important in the quality control system of TCH.The development of the TCH identification techniques has improved the quality evaluation level of TCH.By so far,the TCH identification techniques have been developed from traditional pharmacognostic identification methods based on cell and sub-cell to genetic DNA(gDNA) level.It is a tendency to use DNA microarray to identify the species of TCH in recent years.In this review,a new method called suppression subtraction hybridization and DNA array(SSH-array) was introduced,which combined the SSH technique with microarray hybridization based on nylon film.Five plants from Dendrobium Sw.were used as experimental materials.Firstly the differential gDNA fragments between two species by SSH-array were obtained and then the diferential gDNA fragments with the microarrays made up of multiple whole genomes from several species were hybridized to screen the uniqued gDNA fragments for one species.The screened unique gDNA fragment can be used as species-specific probes for identifying the species.Based on the method,a procedure for species identification of TCH was established with gDNA microarray and its applied prospects were discussed.