Multiplexed Detection of Fungal Nucleic Acid Signatures

Mara R. Diaz1, Sherry A. Dunbar2, James W. Jacobson2

1 University of Miami, Rosenstiel School of Marine and Atmospheric Science, Miami, Florida, 2 Luminex Corporation, Austin, Texas
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.9
DOI:  10.1002/0471142956.cy1309s44
Online Posting Date:  April, 2008
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Abstract

Diagnoses of opportunistic mycotic infections constitute an increasing clinical problem. Conventional diagnostic tests are time consuming and lack specificity and sensitivity for accurate and timely prognoses. This unit provides a comprehensive description of a fungal detection method that combines nucleic acid signatures with flow cytometry. The multiplexed assay, which uses xMAP technology, consists of unique fluorescent microspheres covalently bound to species‐specific fungal oligonucleotide probes. In the presence of the complementary target sequence, the probe hybridizes to its biotinylated target. Quantification of the reaction is based on the fluorescence signal of the reporter molecule that binds to the biotin moieties of the target. The assay can be expanded to include other microorganisms and has the capability to simultaneously test 100 different fungal probes per tube/well. The speed, flexibility in design, and high‐throughput capability makes this assay an attractive diagnostic tool for fungal infections and other related maladies. Curr. Protocol. Cytom. 44:13.9.1‐13.9.21. © 2008 by John Wiley & Sons, Inc.

Keywords: fungi; yeast; Luminex; bead suspension array; nucleic acid; hybridization; multiplex

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Direct Hybridization Assay
  • Support Protocol 1: Hybridization Optimization
  • Support Protocol 2: Nucleic Acid Extraction
  • Support Protocol 3: PCR Amplification
  • Support Protocol 4: Microsphere Coupling
  • Support Protocol 5: Data Analysis
  • Reagents and Solutions
  • Commentary
  • LIterature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Direct Hybridization Assay

  Materials
  • Coupled carboxylated microspheres (see for probe design; see protocol 5 for probe coupling)
  • 1.5× and 1× TMAC solution (see recipe)
  • TE buffer, pH 8 ( appendix 2A; or purchase as 100× solution from Sigma)
  • Biotinylated amplicon samples ( protocol 4)
  • 1 mg/ml streptavidin‐R‐phycoerythrin (SAPE; Molecular Probes)
  • 96‐well plates and sealing cover
  • 1.5‐ml microcentrifuge tubes
  • Vortex
  • Sonicator bath
  • 1‐ to 1000‐µl micropipettors
  • Thermal cycler or heating block
  • Centrifuge with rotor adapted to 96‐well plates
  • Luminex 100 or 200 flow cytometer (or equivalent)

Support Protocol 1: Hybridization Optimization

  Materials
  • Fungal cell cultures, in suspension or on agar
  • RNase‐free water (e.g., USB)
  • Lysing enzyme solution: 10 mg lysing enzyme from Trichoderma harzianum (Sigma)/ml lysing buffer (see recipe)
  • 95% (v/v) ethanol
  • QIAamp DNeasy tissue culture buffers (ATL, AL, AW, AW2, AE; Qiagen): AE buffer prewarmed to 70°C
  • 3‐ or 4‐mm bacteriological loop
  • 2‐ml microcentrifuge tubes
  • 37°C, 55°C, and 70°C water baths or heating blocks
  • Centrifuge
  • Spin columns (included in DNeasy kit, Qiagen)

Support Protocol 2: Nucleic Acid Extraction

  Materials
  • Genomic DNA (see )
  • Qiagen HotStar Taq Master Mix Kit (Qiagen)
  • 10 pmol/µl forward and biotinylated reverse primer (see Fig. and Table 13.9.2)
  • RNase‐free water (e.g., USB)
  • PCR tubes or 96‐well plates with sealing covers
  • Thermal cycler

Support Protocol 3: PCR Amplification

  Materials
  • Carboxylated microspheres (Mirai Bio)
  • 100 µM 5′C‐12‐amino modified oligo (capture probe; IDT technologies), Tris‐ and azide‐free, desalted
  • Coupling buffer (see recipe)
  • 10 mg/ml 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC; Pierce)
  • TE, pH 8 ( appendix 2A; or purchase as 100× solution from Sigma)
  • 0.02% (v/v) Tween 20: sterilized by passing through a 0.2‐µm filter and stored up to 6 months at room temperature
  • 0.01% (v/v) SDS: prepared using 10% (w/v) SDS, sterilized by passing through a 0.2‐µm filter, and stored up to 6 months at room temperature
  • Bath sonicator
  • 1.5‐ml copolymer microcentrifuge tubes (USA Scientific, cat. no. 1415‐2500)
  • Centrifuge
  • Hemacytometer
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Figures

Videos

Literature Cited

LIterature Cited
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