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Comparison of Two Commercially Available qPCR Kits for the Detection of Candida auris.

ABSTRACT: Candida auris is an emerging pathogen with resistance to many commonly used antifungal agents. Infections with C. auris require rapid and reliable detection methods to initiate successful medical treatment and contain hospital outbreaks. Conventional identification methods are prone to errors and can lead to misidentifications. PCR-based assays, in turn, can provide reliable results with low turnaround times. However, only limited data are available on the performance of commercially available assays for C. auris detection. In the present study, the two commercially available PCR assays AurisID (OLM, Newcastle Upon Tyne, UK) and Fungiplex Candida Auris RUO Real-Time PCR (Bruker, Bremen, Germany) were challenged with 29 C. auris isolates from all five clades and eight other Candida species as controls. AurisID reliably detected C. auris with a limit of detection (LoD) of 1 genome copies/reaction. However, false positive results were obtained with high DNA amounts of the closely related species C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. The Fungiplex Candida Auris RUO Real-Time PCR kit detected C. auris with an LoD of 9 copies/reaction. No false positive results were obtained with this assay. In addition, C. auris could also be detected in human blood samples spiked with pure fungal cultures by both kits. In summary, both kits could detect C. auris-DNA at low DNA concentrations but differed slightly in their limits of detection and specificity.

SUBMITTER: Sattler J

PROVIDER: S-EPMC7926799 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Comparison of Two Commercially Available qPCR Kits for the Detection of Candida auris.

Sattler Janko J Noster Janina J Brunke Anne A Plum Georg G Wiegel Pia P Kurzai Oliver O Meis Jacques F JF Hamprecht Axel A

Journal of fungi (Basel, Switzerland) 20210222 2

Candida auris is an emerging pathogen with resistance to many commonly used antifungal agents. Infections with C. auris require rapid and reliable detection methods to initiate successful medical treatment and contain hospital outbreaks. Conventional identification methods are prone to errors and can lead to misidentifications. PCR-based assays, in turn, can provide reliable results with low turnaround times. However, only limited data are available on the performance of commercial ...[more]

PMID: 33671676

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Project description:BACKGROUND: Describing and evaluating miRNA inventories with Next Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do not only contain abundant ribosomal RNAs but are also enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris, an important but small (500 ?m in length) monogenean pathogen of Norwegian Atlantic salmon (Salmo salar). FINDINGS: We evaluated 1 salt precipitation method (MasterPure™ Complete RNA Purification Kit, Epicentre), 2 Phenol based extraction methods (mirVana Kit, Ambion, and Trizol Plus Kit, Invitrogen), 1 paramagnetic bead extraction method (RNA Tissue kit, GeneMole) and 2 purification methods based on spin column chromatography using a proprietary resin as separation matrix (Phenol-free Total RNA Purification Kit, Amresco, and ZR MicroPrep Kit, Zymo Research). The quality of the extractions from 1, 10 and 100 individuals, respectively, was assessed in terms of totalRNA yield, RNA integrity, and smallRNA and miRNA yield. The 6 RNA extraction methods yielded considerably different total RNA extracts, with striking differences in low molecular weight RNA yield. The Phenol-free Total RNA Purification Kit (Amresco) showed the highest totalRNA yield, but the best miRNA/totalRNA ratio was obtained with the ZR MicroPrep Kit (Zymo Research). It was not possible to extract electrophoretically detectable miRNAs from Gyrodactylus salaris with the RNA Tissue Kit (GeneMole) or the Trizol Plus Kit (Invitrogen). CONCLUSIONS: We present an optimized extraction protocol for single and small numbers of Gyrodactylus salaris from infected Atlantic salmon that delivers a totalRNA yield suitable for downstream next generation sequencing analyses of miRNA. Two of the six tested totalRNA kits/methods were not suitable for the extraction of miRNAs from Gyrodactylus salaris.

Dataset Information

Comparison of Two Commercially Available qPCR Kits for the Detection of Candida auris.

Publications

Comparison of Two Commercially Available qPCR Kits for the Detection of <i>Candida auris</i>.

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