Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR

Abstract

A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Furthermore, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of Aspergillus, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.

High-Throughput Identification and Quantification of Candida Species Using High Resolution Derivative Melt Analysis of Panfungal Amplicons

“…There are a number of publications reporting a variety of primers for this approach, with widely varying levels of rigor in their validation. [55][56][57][58][59][60][61][62] In general, this approach has the disadvantage that multiple assays have to be run on each sample, adding cost and labor. Multiplexing is a possible alternative, but this is widely associated with reduced sensitivity.…”

A new multiplex PCR for species-specific diagnosis of human candidiasis

“…Se han determinado parámetros similares con esta misma herramienta computacional usando secuencias cebadoras previamente descritas en ensayos de PCR para la identificación de especies de Candida (19)(20)(21)(22)(23). Estos resultados se incluyeron en el cuadro 1 con fines de comparación.…”

“…Las secuencias de los oligonucleótidos utilizados se optimizaron para garantizar la sensibilidad y la especificidad con base en todos los parámetros de diseño termodinámicos y de resolución descritos por García, et al (16), y por Allawi, et al (17), mediante la evaluación de las secuencias diseñadas con el algoritmo computacional Mult-PSOS (16). Los análisis comparativos por simulación computacional de los parámetros físicos y estructurales de las secuencias de la PCR múltiple en este estudio se compararon con las secuencias reportadas con otros métodos (19)(20)(21)(22)(23). Como queda evidenciado en el cuadro 1, los resultados predijeron una mayor eficiencia de nuestro ensayo, pues las secuencias diseñadas presentaron: a) una menor formación de estructuras secundarias (dímeros, bucles internos, bucles en horquilla y bucles en bulto), las cuales afectan negativamente la sensibilidad de la corrida; b) un mejor ajuste de las temperaturas de fusión (en su mayoría superiores a los 50 °C y con menores diferencias entre el conjunto de cebadores) y del porcentaje de GC (entre 40 y 60 %), y c) una diferencia entre el tamaño de amplicones que permitió su diferenciación en geles comunes de agarosa (cercana a las 50 pb).…”

Characterization of the Same Mutations in FCA1 Gene Associated With 5-FC Resistance of Candida albicans

“…Then, total genomic DNA templates of these isolates were extracted using the HP Fungal DNA Kit (Omega bio-tec Ltd., USA) based on the manufacture's instruction. PCR reaction was performed with primers (11)(12)(13)…”