Analysis of Polymorphic Microsatellite Markers for Typing Penicillium marneffei Isolates

Abstract

Penicillium marneffei is an emerging opportunistic dimorphic fungal pathogen that is endemic in Southeast Asia. A typing method based on the analysis of size polymorphisms in microsatellite loci was investigated. Three loci available from the GenBank database were identified to harbor microsatellites. PCR primers flanking the microsatellite repeats were designed with one primer in the set fluorescently labeled. PCR products were then sized by automated capillary electrophoresis. As expected for a haploid fungus, a single band was observed for each microsatellite locus for all isolates. Polymorphic microsatellite marker (PMM) analysis detected a total of 22 different allelic types for 35 isolates of P. marneffei with a high discriminatory power (D ‫؍‬ 0.956). Microsatellites I, II, and III detected 14, 10, and 7 alleles, respectively. The reproducibility of length polymorphisms was confirmed by using different DNA preparations from the same isolate or by repeated runs from the same DNA preparation. PMM profiles for eight isolates passaged in vitro for 7 to 8 weeks were identical to the original culture, demonstrating short-term stability and reproducibility. PCR products were not observed for other dimorphic fungi or human DNA. Comparison of allelic frequencies in isolates obtained from China and Thailand identified distinct allele combinations, suggesting the potential geographic isolation of populations. Due to the high discriminatory power, reproducibility, and potential for high throughput, PMM analysis may provide a good typing method for epidemiologic and surveillance investigations of P. marneffei.

Nucleotide Sequence-Based Analysis for Determining the Molecular Epidemiology of Penicillium marneffei

“…Genomic DNA was purified from mycelial phase cells as described by Lasker and Ran (30). DNA purified from isolates subcultured in vitro for 7 to 8 weeks was described previously (30).…”

“…The in vitro reproducibility of MLST analysis was evaluated by analysis of DNA sequences obtained from DNA purified from mycelial cells grown in broth for 7 to 8 weeks by passing the cells in fresh broth every 2 to 3 days (30). In all cases no differences were detected between the nucleotide sequences obtained from the original DNA preparations from the nucleotide sequences obtained from the DNA preparations from cultures transferred in vitro.…”

“…Isolates obtained from humans and bamboo rats were observed to share a common genotype; however, the route of infection and transmission to humans has not been determined, although aerosolization of conidia has been proposed as the most likely mechanism (21,54). Recently, typing using polymorphic microsatellite markers proposed geographic differences in population and localized dispersion of strains, but no clear epidemiologic link was observed between environmental and clinical isolates (17,30).…”

Diversidade genética e funcional de leveduras presentes em solos de mineração e áreas do entorno

“…A utilização desta técnica tem crescido significativamente e, dependendo do primer utilizado e das bandas analisadas, tem poder discriminatório para diferenciar linhagens ou espécies (Lasker & Ran, 2004 Para identificação mais segura, atualmente são utilizados em conjunto a taxonomia morfológica e molecular, conhecido por Taxonomia Polifásica onde todos os caracteres mencionados são levados em consideração para a identificação de leveduras (Libkind et al, 2003;Sampaio et al, 2001;Sampaio, 2002;Duarte et al, 2013;Wuczkowski & Prillinger, 2004).…”

“…Muitos trabalhos vem utilizando a técnica de MSP-PCR para agrupamento de isolados recuperados de amostras ambientais, como uma ferramenta para diminuir a quantidade de isolados que serão sequenciados, uma vez que tem poder discriminatório para diferenciar linhagens ou espécies (Lasker & Ran, 2004) Embora a análise feita pelo programa Bionumerics não tenha sido conclusiva. A técnica de MSP-PCR pode, em alguns casos, gerar perfis diferentes para membros da mesma espécie, que pode ser indicativo de populações diferentes dentro das espécies , porém continua sendo uma abordagem interessante em estudos de ecologia de leveduras, pois na maioria dos casos gera dados rápidos e acurados.…”