Development and Analysis of qPCR for the Identification of Arthroconidial Yeasts of the Genus Magnusiomyces

TY - JOUR

T1 - Development and Analysis of qPCR for the Identification of Arthroconidial Yeasts of the Genus Magnusiomyces

AU - Kaplan, Engin

AU - Aktaş, Deniz

AU - Döğen, Aylin

AU - Hilmioğlu-Polat, Süleyha

AU - Gümral, Ramazan

AU - Hagen, Ferry

AU - Ilkit, Macit

AU - de Hoog, G Sybren

PY - 2021/3

Y1 - 2021/3

N2 - The arthroconidial yeasts Magnusiomyces capitatus and M. clavatus are emerging opportunistic pulmonary pathogens. They are closely related and difficult to distinguish based on morphological and physiological traits. We applied an SYBR® green-based quantitative PCR (qPCR) assay to identify the species. We analyzed 30 reference strains originating from clinical and environmental sources by targeting the Rpb2 gene encoding the second largest subunit of RNA polymerase II. The qPCR assays were tested by direct identification of M. capitatus and M. clavatus in spiked sputum and household dishwasher swabs, respectively, as models for clinical and environmental samples. The assays were proved to be reliable for species-level identification of both species, with 100% sensitivity and 100% specificity, lowest inter-assay deviations (RSDr ≤ 1.65%, R2 values >0.99), detection limit of 10 theoretical copy number of target DNA, and detection cell limit of ≥5000 yeast cells from spiked sputum samples. The developed qPCR assay is a practical molecular approach for the detection of M. capitatus and M. clavatus that can be used as a stand-alone assay or in conjunction with culture-dependent approaches.

AB - The arthroconidial yeasts Magnusiomyces capitatus and M. clavatus are emerging opportunistic pulmonary pathogens. They are closely related and difficult to distinguish based on morphological and physiological traits. We applied an SYBR® green-based quantitative PCR (qPCR) assay to identify the species. We analyzed 30 reference strains originating from clinical and environmental sources by targeting the Rpb2 gene encoding the second largest subunit of RNA polymerase II. The qPCR assays were tested by direct identification of M. capitatus and M. clavatus in spiked sputum and household dishwasher swabs, respectively, as models for clinical and environmental samples. The assays were proved to be reliable for species-level identification of both species, with 100% sensitivity and 100% specificity, lowest inter-assay deviations (RSDr ≤ 1.65%, R2 values >0.99), detection limit of 10 theoretical copy number of target DNA, and detection cell limit of ≥5000 yeast cells from spiked sputum samples. The developed qPCR assay is a practical molecular approach for the detection of M. capitatus and M. clavatus that can be used as a stand-alone assay or in conjunction with culture-dependent approaches.

U2 - 10.1007/s11046-020-00510-4

DO - 10.1007/s11046-020-00510-4

M3 - Article

C2 - 33392857

SN - 0301-486X

VL - 186

SP - 41

EP - 51

JO - Mycopathologia

JF - Mycopathologia

IS - 1

ER -