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Rapid detection and identification of mucormycetes from culture and tissue samples by use of high-resolution melt analysis.

ABSTRACT: We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.

SUBMITTER: Hrncirova K

PROVIDER: S-EPMC2937726 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Rapid detection and identification of mucormycetes from culture and tissue samples by use of high-resolution melt analysis.

Hrncirova Kristyna K Lengerova Martina M Kocmanova Iva I Racil Zdenek Z Volfova Pavlina P Palousova Dita D Moulis Mojmir M Weinbergerova Barbora B Winterova Jana J Toskova Martina M Pospisilova Sarka S Mayer Jiri J

Journal of clinical microbiology 20100630 9

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics. ...[more]

PMID: 20592150

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Project description:Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen). The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence.

Dataset Information

Rapid detection and identification of mucormycetes from culture and tissue samples by use of high-resolution melt analysis.

Publications

Rapid detection and identification of mucormycetes from culture and tissue samples by use of high-resolution melt analysis.

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