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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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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Basic Protocol 1: Direct Hybridization Assay

  • 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

  • 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

  • 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

  • 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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Literature Cited

LIterature Cited
   Abou‐ela, F., Koh, D., Tinoco, I. Jr., and Martin, F.J. 1985. Base‐base mismatches of Thermodynamics of double helix formation for dCA3xA3G dCT3yT3G (x,y = A, C, G, T). Nucl. Acids Res. 13: 3944‐3948.
   Anaissie, E. 2000. Phaeohyphomycosis: New perspectives on diagnostics and treatment. Program and Abstracts of the 38th Annual Meeting of the Infectious Diseases Society of America, abstr. S66.
   Armstrong, B., Stewart, M., and Mazumder, A. 2000. Suspension arrays for high throughput, multiplexed single nucleotide polymorphism genotyping. Cytometry 40: 102‐108.
   Baums, I.B., Goodwin, K.D., Kiesling, T., Wanless, D., Diaz, M.R., and Fell, J.W. 2007. Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand. Mar. Pollut. Bull. 54: 521‐536.
   Belkhiri, A., Buchko, J., and Klassen, G.R. 1992. The 5S ribosomal RNA gene in Pythium species: Two different genomic locations. Mol. Biol. Evol. 9: 1089‐1102.
   Bovers, M., Diaz, M.R., Hagen, F., Spanjaard, L., Duim, B., Visser, C.E., Hoogveld, H.L., Scharringa, J., Hoepelman, I.M., Fell, J.W., and Boekhout, T. 2007. Identification of genotypically diverse Cryptococcus neoformans and Cryptococcus gattii isolates using Luminex xMAP technology. J. Clin Microbiol. 45: 1874‐1883.
   Cassidy, J.R. and Pukkila, P.J. 1987. Inversion of Inversion of 5S ribosomal RNA genes within the genus. Coprinus. Curr. Genet. 12: 33‐36.
   Cheng, H.R. and Jiang, N. 2006. Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol. Lett. 28: 55‐59.
   de Hoog, G.S., Guarro, J., Gené, J., and Figueras, M.J. 2000. Atlas of Clinical Fungi. 2nd ed. p. 1124. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands/Universitat Rovirai Virgili, Reus, Spain.
   Diaz, M.R. and Fell, J.W. 2004. High‐throughput detection of pathogenic yeasts in the genus Trichosporon. J. Clin. Microbiol. 42: 3696‐3706.
   Diaz, M.R. and Fell, J.W. 2005. Use of a suspension array for rapid identification of the varieties and genotypes of the Cryptococcus neoformans species complex. J. Clin. Microbiol. 43: 3662‐3672.
   Diaz, M. R., Boekhout, T., Theelen, B., Bovers, M., Cabañes, F.J., and Fell, J.W. 2006. Microcoding and flow cytometry as identification system for Malassezia species. J. Med. Microbiol. 55: 1197‐1209.
   Dunbar, S.A. and Jacobson, J.W. 2007. Quantitative, multiplexed detection of Salmonella and other pathogens by Luminex xMAP suspension array. Methods. Mol. Biol. 394: 1‐19.
   Dunbar, S.A., Vander Zee, C.A., Oliver, K.G., Karem, K.L., and Jacobson, J.W. 2003. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMap system. J. Microbiol. Meth. 53: 245‐252.
   Fulton, R., McDade, R., Smith, P., Kienker, L., and Kettman, J. 1997. Advanced multiplexed analysis with the FlowMetrix system. Clin. Chem. 43: 1749‐1756.
   Hacia, J.G. 1999. Resequencing and mutational analysis using oligonucleotide microarrays. Nature Genet. 21: 42‐47.
   Jacobson, J.W., Oliver, K.G., Weiss, C., and Kettman, J. 2006. Analysis of Individual Data from Bead‐Based Assays (‘‘Bead Arrays’’). Cytometry A. 69A: 384‐390.
   Koehler, R.T. and Peyret, N. 2005. Effects of DNA secondary structure on nucleotide probe binding efficiency. Comput Biol. Chem. 29: 393‐397.
   Lockhart, D.J., Dong, H., Byrne, M.C., Folletie, M.T., Gallo, M.V., Chee, M.S., Mittman, M., Wang, C., Kobayashi, M., Horton, H., and Brown, E.L. 1996. Expression monitoring by hybridization to high‐density oligonucleotide arrays. Nat. Biotechnol. 14: 1675‐1680.
   Maskos, U. and Southern, E.M. 1993. A study of oligonucleotide reassociation using large arrays of oligonucleotides synthesized on a large support. Nucl. Acids Res. 21: 4663‐4669.
   McNamara, D.T., Kasehagen, L.J., Grimberg, B.T., Cole‐Tobian, J., Collins, W.E., and Zimmerman, P.A. 2006. Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent microsphere‐based assay. Am. J. Trop. Med. Hyg. 74: 413‐421.
   Page, B.T. and Kurtzman, C.P. 2005. Rapid identification of Candida species and other clinically important yeast species by flow cytometry. J. Clin. Microbiol. 43: 4507‐4514.
   Syn, C.K. and Swarup, S. 2000. A scalable protocol for the isolation of large‐sized genomic DNA within an hour from several bacteria. Anal. Biochem. 278: 86‐90.
   Taylor, J.D., Briley, D., Nguyen, Q., Long, K., Iannone, M.A., Li, M.S., Ye, F., Afshari, A., Lai, E., Wagner, M., Chen, J., and Weiner, M.P. 2001. Flow cytometric platform for high‐throughput single nucleotide polymorphism analysis. Biotechniques 30: 661‐669.
   Williams, J.C., Case‐Green, S.C., Mir, S.C, and Southern, E.M. 1994. Studies of oligonucleotide interactions by hybridization to arrays: the influence of dangling ends on duplex yield. Nucl. Acids Res. 22: 1365‐1367.
   Wilson, K. 1990. Preparation of genomic DNA from bacteria. Curr. Protoc. Mol. Biol. 27: 2.4.1‐2.4.5.
   Wilson, W.J., Erler, A.M., Nasarabadi, S.L., Skowronski, E.W., and Imbro, P.M. 2005. A multiplexed PCR‐coupled liquid bead array for the simultaneous detection of four biothreat agents. Mol. Cell. Probe. 19: 137‐144.
   Ye, F., Li, M.S., Taylor, J.D., Nguyen, Q., Colton, H.M, Casey, W.M., Wagner, M., Weiner, M.P., and Chen, J. 2001. Fluorescent microsphere‐based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification. Hum. Mutat. 17: 305‐316.
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