Selection of Polymorphic Patterns Obtained by RAPD-PCR through Qualitative and Quantitative Analyses to Differentiate Aspergillus fumigatus.

Omar E Valencia-Ledezma,Carlos A Castro-Fuentes,María Guadalupe Frías-De-León,María Del Rocío Reyes-Montes,Esperanza Duarte-Escalante

doi:10.3390/jof8030296

Abstract

The objective of this work was to use the random amplification of the polymorphic DNA–polymerase chain reaction (RAPD-PCR) technique to select polymorphic patterns through qualitative and quantitative analyses to differentiate the species A. flavus, A. fumigatus, A. niger and A. tubingensis. Twenty-seven Aspergillus isolates from different species were typified using phenotypic (macro- and micromorphology) and genotypic (partial BenA gene sequencing) methods. Thirty-four primers were used to obtain polymorphic patterns, and with these a qualitative analysis was performed to select the primers that presented species-specific patterns to distinguish each species. For the quantitative selection, a database was built from the polymorphic patterns and used for the construction of logistic regression models; later, the model that presented the highest value of sensitivity against specificity was evaluated through ROC curves. The qualitative selection showed that the primers OPA-19, P54, 1253 and OPA-02 could differentiate the species. A quantitative analysis was carried out through logistic regression, whereby a species-specific correlation of sensitivity and specificity greater than 90% was obtained for the primers: OPC-06 with a 96.32% match to A. flavus; OPF-01 with a 100% match to A. fumigatus; OPG-13 with a 98.01% match to A. tubingensis; and OPF-07 with a 99.71% match to A. niger. The primer OPF-01 discriminated the four species as well as closely related species. The quantitative methods using the selected primers allowed discrimination between species and showed their usefulness for genotyping some of the species of medical relevance belonging to the genus Aspergillus.

Highlights

Introduction published maps and institutional affilWithin the genus Aspergillus, more than 400 species are recognized [1], and the most important ones from a pathogenic point of view are included in the sections Fumigati, Flavi and Nigri, representing more than 95% of the pathogenic Aspergillus species
The increase in immunocompromised patients associated with invasive aspergillosis, as well as new Aspergillus spp., points to the need to identify the causative agents at the species level, and a rapid and reliable diagnosis is necessary, so the objective of this work was to select polymorphic patterns obtained by RAPD-PCR through qualitative and quantitative analyses to differentiate the species A. flavus, A. fumigatus, A. niger and
The RAPD-PCR technique was used to obtain polymorphic patterns and, based on these, perform qualitative and quantitative selections that would allow the identification of specific primers for the species A. flavus, A. fumigatus and A. tubingensis

Summary

Introduction

Within the genus Aspergillus, more than 400 species are recognized [1], and the most important ones from a pathogenic point of view are included in the sections Fumigati, Flavi and Nigri, representing more than 95% of the pathogenic Aspergillus species. It is important to mention that, in the last two decades, there has been a significant increase in invasive fungal infections, invasive aspergillosis (IA) [2]. Among the most relevant opportunistic pathogenic species within the Fumigati section are A. fumigatus, A. lentulus, A. fumigatiaffinis, A. fumisynnematus, A. novofumigatus and A. laciniosa [3]. The diagnosis of invasive aspergillosis is challenging, in immunocompromised patients [6]. The signs and symptoms are nonspecific, colonization is difficult to distinguish from an invasive disease, blood cultures are commonly negative and patients are often unable to undergo invasive diagnostic procedures [7]

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