Infectious Diseases Testing

Gregory L. Blakey1, Ruth Ann Luna2, James Versalovic2

1 Baylor College of Medicine, Houston, Texas, 2 Baylor College of Medicine and Texas Children's Hospital, Houston, Texas
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.18
DOI:  10.1002/0471142905.hg0918s47
Online Posting Date:  November, 2005
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Abstract

Molecular methods have been applied widely for the diagnosis of infectious diseases. Beginning with solution hybridization in the early 1990s, multiple methods for nucleic acid hybridization and amplification have been introduced into the laboratory for the identification and characterization of microbial pathogens. This unit contains examples of several basic approaches for microbial detection or characterization in the laboratory. Methods in this chapter include automated nucleic acid extraction, direct detection of microbial pathogens, and characterization of pathogens by DNA sequencing or typing. Any of these methods could be customized for the characterization of bacteria, fungi, parasites, or viruses.

Keywords: automated DNA extraction; end‐point PCR; real‐time PCR; pyrosequencing; real‐time DNA sequencing; rep‐PCR

     
 
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Table of Contents

  • Basic Protocol 1: HIV‐1 Detection and Quantification by End‐Point RT‐PCR (COBAS AMPLICOR HIV‐1 MONITOR Test)
  • Support Protocol 1: Automated Viral Nucleic Acid Extraction (For HIV‐1 Detection and Quantification)
  • Basic Protocol 2: mecA Detection by Real‐Time Fluorescent PCR
  • Support Protocol 2: Automated Bacterial DNA Extraction
  • Basic Protocol 3: Bacterial Identification by Real‐Time Sequencing of 16S Ribosomal DNA
  • Basic Protocol 4: Bacterial Strain Identification by REP‐PCR (DiversiLab)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: HIV‐1 Detection and Quantification by End‐Point RT‐PCR (COBAS AMPLICOR HIV‐1 MONITOR Test)

  Materials
  • Purified RNA in Roche MagNA Pure elution buffer (50 µl per sample isolated from plasma; see protocol 2)
  • COBAS AMPLICOR HIV‐1 MONITOR kit v1.5 (Roche Diagnostics; store at 4°C) containing:
    • HIV MONITOR master mix
    • HIV MONITOR manganese solution
    • IM PS1
    • IQ PS1
    • SB
    • AD3 cassette
    • DN4 cassette
    • CN4 cassette
    • 10× wash buffer
    • High and low positive controls
  • COBAS AMPLICOR analyzer (Roche Diagnostics)
  • COBAS AMPLICOR A‐ring tubes and D‐cup disposables (Roche Diagnostics)
  • Sealable plastic bag

Support Protocol 1: Automated Viral Nucleic Acid Extraction (For HIV‐1 Detection and Quantification)

  Materials
  • Blood or 200 µl plasma
  • Nuclease‐free water
  • MagNA Pure LC total nucleic acid isolation kit (Roche Diagnostics) including:
    • Elution buffer
    • Proteinase K
    • Wash buffer I
    • Wash buffer II
    • Wash buffer III
    • Lysis/binding buffer
    • Magnetic glass particle suspension
    • QS standard from COBAS AMPLICOR HIV‐1 MONITOR kit v1.5 (Roche Diagnostics)
  • MagNA Pure LC (Roche Diagnostics)
  • MagNA Pure LC disposables (Roche Diagnostics):
    • Sample cartridges
    • Reagent tubs (small, medium‐20, medium‐30, and large)
    • Reagent tub lids (small, medium, and large)
    • Processing cartridges
    • Reaction tips (small and large)
    • Tip stands
    • Drop catchers
    • Cartridge seals
    • Waste bottles
CAUTION: Avoid contact with wash buffer I (contains guanidine hydrochloride) and lysis/binding buffer (contains guanidine isothiocyanate). Avoid adding bleach to lysis/binding buffer (produces a toxic gas).NOTE: Heat MagNA Pure LC isolation kit solutions up to 37°C to dissolve any precipitates. Store MagNA Pure LC isolation kit contents at 15° to 25°C.

Basic Protocol 2: mecA Detection by Real‐Time Fluorescent PCR

  Materials
  • Extracted genomic DNA samples, LightCycler mecA template DNA (positive control plasmid), bacterial positive control, and bacterial negative control (see protocol 4)
  • 10× LightCycler fast‐start DNA master hybridization probes containing:
    • FastStart Taq DNA polymerase
    • Reaction buffer
    • dNTPs
    • 10 mM MgCl 2
  • PCR‐grade H 2O
  • 25 mM MgCl 2
  • 10× LightCycler mecA primers/hybridization probes mix:
  • mecA probe set
    • Probe 1 (label: LightCycler‐Red 640)
    • Probe 2 (label: fluorescein)
  • Internal control probe set
    • Probe 3 (label: LightCycler‐Red 705)
    • Probe 4 (label: fluorescein)
  • LightCycler mecA recovery template DNA (internal control plasmid)
  • LightCycler color compensation set
  • LightCycler 1.0
  • LightCycler capillaries and stoppers
  • LightCycler carousel
  • 2‐ml tube rotor for conventional centrifuge or LC carousel centrifuge
  • LightCycler software
NOTE: Store all reagents at −15° to −25°C. Avoid repeated freeze‐thaw cycles. Avoid light exposure of fluorescently‐labeled probes.

Support Protocol 2: Automated Bacterial DNA Extraction

  Materials
  • 0.75% dithiothreitol (DTT)
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • MagNA Pure LC DNA Isolation Kit III (Roche Diagnostics) including:
    • Elution buffer
    • Proteinase K
    • Bacteria lysis buffer
    • Wash buffer I
    • Wash buffer II
    • Wash buffer III
    • Lysis/binding buffer
    • Magnetic glass particles suspension
  • 37°C heating block
  • MagNA Pure LC (Roche Diagnostics)
  • Vortex mixer
  • MagNA Pure LC disposables (Roche Diagnostics):
    • Sample cartridges
    • Reagent tubs (small, medium‐20, medium‐30, and large)
    • Reagent tub lids (small, medium, and large)
    • Processing cartridges
    • Reaction tips (small and large)
    • Tip stands
    • Drop catchers
    • Cartridge seals
    • Waste bottles
CAUTION: Avoid contact with wash buffer I (contains guanidine hydrochloride) and lysis/binding buffer (contains guanidine isothiocyanate). Avoid adding bleach to lysis/binding buffer (produces a toxic gas).NOTE: Heat MagNA Pure LC isolation kit solutions up to 37°C to dissolve any precipitates. Store MagNA Pure LC isolation kit contents at 15° to 25°C.

Basic Protocol 3: Bacterial Identification by Real‐Time Sequencing of 16S Ribosomal DNA

  Materials
  • PCR‐grade water
  • dNTPs (GeneAmp, Applied Biosystems)
  • 25 mM MgCl 2 (Applied Biosystems)
  • 10× PCR buffer (GeneAmp, Applied Biosystems)
  • V1 primers (10 µM for PCR; 3 µM for sequencing):
  • AmpliTaq Gold LD DNA polymerase (Applied Biosystems)
  • V3 primers (10 µM for PCR; 3 µM for sequencing):
  • 10 ng/µl purified genomic bacterial DNA
  • Positive control DNA
  • Binding buffer (Biotage)
  • Streptavidin‐Sepharose HP (Amersham Biosciences)
  • 70% ethanol
  • Washing buffer (Biotage)
  • Denaturation solution (Biotage)
  • Annealing buffer (Biotage)
  • PSQ 96 SQA reagent kit (Biotage) containing:
    • Enzyme mixture
    • Substrate mixture
    • dATPαS, dCTP, dGTP, and dTTP
  • 0.2‐ml PCR tubes
  • Vortex mixer
  • Conventional thermal cycler
  • 96‐well plate
  • Microtiter plate shaker/mixer
  • Reagent troughs
  • Vacuum prep tool (Biotage)
  • PSQ 96 sequencing plates (Biotage)
  • 80°C heating block
  • Pyrosequencer (Biotage)
  • PSQ 96 reagent cartridge (Biotage)
NOTE: Store the enzyme mixture, substrate mixture, and all PCR reagents at −20°C. Store the remaining reagents except ethanol and water at 4°C.

Basic Protocol 4: Bacterial Strain Identification by REP‐PCR (DiversiLab)

  Materials
  • DiversiLab rep‐PCR Kit (Bacterial Barcodes) for PCR containing:
    • Rep‐PCR MM1
    • Organism‐specific primer mix
    • Positive control C3
    • Negative control
  • 10× PCR buffer with 15 mM MgCl 2 (GeneAmp, Applied Biosystems)
  • AmpliTaq DNA polymerase (Applied Biosystems)
  • 25 ng/µl purified genomic DNA
  • DiversiLab rep‐PCR kit (Bacterial Barcodes) for microfluidic separation containing:
    • DNA gel matrix
    • DNA dye concentrate
    • Spin filters
    • DNA ladder
    • DNA marker
    • DNA chips
  • 1.5‐ml microcentrifuge tubes
  • 0.2‐ml PCR tubes
  • Thermal cycler
  • BioAnalyzer model 2100 with chip priming station and vortex mixer (Agilent Technologies) and LabChip software
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Figures

Videos

Literature Cited

   Cole, J.R., Chai, B., Marsh, T.L., Farris, R.J. Wang, Q., Kulam, S.A., Chandra, S., McGarrell, D.M., Schmidt, T.M., Garrity, G.M., and Tiedje, J.M. 2003. The Ribosomal Database Project (RDP‐II): Previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucl. Acids Res. 31:442‐443.
   Drancourt, M., Berger, P., and Raoult, D. 2004. Systematic 16S rRNA gene sequencing of atypical clinical isolates identified 27 new bacterial species associated with humans. J. Clin. Microbiol. 42:2197–2202.
   Gharizadeh, B., Kalantari, M., Garcia, C.A., Johansson, B., and Nyrén, P. 2001. Typing of human papillomavirus by Pyrosequencing. Lab. Invest. 81:673‐679.
   Gharizadeh, B., Norberg, E., Löffler, J., Jalal, S., Tollemar, J., Einsele, H., Klingspor, L., and Nyrén, P. 2004. Identification of medically important fungi by the Pyrosequencing technology. Mycoses 47:29‐33.
   Grahn, N., Olofsson, M., Ellnebo‐Svedlund, K., Monstein, H.‐J., and Jonasson, J. 2003. Identification of mixed bacterial DNA contamination in broad‐range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons. FEMS Microbiol. Lett. 219:89‐91.
   Healy, M., Huong, J., Bittner, T., Lising, M., Frye, S., Raza, S., Schrock, R., Manry, J., Renwick, A., Nieto, R., Woods, C., Versalovic, J., and Lupski, J.R. 2005. Microbial DNA typing by automated repetitive‐sequence‐based PCR. J. Clin. Microbiol. 43:199‐207.
   Hill, C.E. and Caliendo, A.M. 2004. Viral load testing. In Molecular Microbiology: Diagnostics Principles and Practice (D.H. Persing, F.C. Tenover, J. Versalovic, Y.‐W. Tang, E.R. Unger, D.A. Relman, and T.J. White, eds.) pp. 475‐487. ASM Press, Washington, DC.
   Hölzl, G., Stöcher, M., Leb, V., Stekel, H., and Berg, J. 2003. Entirely automated quantification of human immunodeficiency virus type 1 (HIV‐1) RNA in plasma by using the ultrasensitive COBAS AMPLICOR HIV‐1 Monitor Test and RNA purification on the MagNA Pure LC instrument. J. Clin. Microbiol. 41:1248‐1251.
   Jonasson, J., Olofsson, M., and Monstein, H.‐J. 2002. Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments. APMIS 110:263‐272.
   Kessler, H.H., M¨hlbauer, G., Stelzl, E., Daghofer, E., Santer, B.I., and Marth, E. 2001. Fully automated nucleic acid extraction: MagNA Pure LC. Clin. Chem. 47:1124‐1126.
   Kolbert, C.P. and Persing, D.H. 1999. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr. Opin. Microbiol. 2:299‐305.
   Knepp, J.H., Geahr, M.A., Forman, M.S., and Valsamakis, A. 2003. Comparison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA. J. Clin. Microbiol. 41:3532‐3236.
   LightCycler MRSA Detection Kit Instruction Manual, version 2. 2003. Roche Diagnostics.
   Lindström, A., Odeberg, J., and Albert, J. 2004. Pyrosequencing for detection of lamivudine‐resistant hepatitis B virus. J. Clin. Microbiol. 42:4788‐4795.
   Loeffler, J., Schmidt, K., Hebart, H., Schumacher, U., and Einsele, H. 2002. Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system. J. Clin. Microbiol. 40:2240‐2243.
   Mackay, I.M. 2004. Real‐time PCR in the microbiology laboratory. Clin. Microbiol. Infect. 10:190‐212.
   MagNA Pure LC DNA Isolation Kit III (Bacteria, Fungi) Instruction Manual. 2004. Roche Diagnostics.
   MagNA Pure LC Total Nucleic Acid Isolation Kit Instruction Manual. 2004. Roche Diagnostics.
   Niesters, H.G.M. 2004. Molecular and diagnostic clinical virology in real time. Clin. Microbiol. Infect. 10:5‐11.
   O'Meara, D., Wilbe, K., Leitner, T., Hejdeman, B., Albert, J., and Lundeberg, J. 2001. Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing. J. Clin. Microbiol. 39:464‐473.
   Peters, I.R., Helps, C.R., Hall, E.J., and Day, M.J. 2004. Real‐time PCR: Considerations for efficient and sensitive assay design. J. Immunol. Methods 286:203‐217.
   Rademaker, J.L.W. and Savelkoul, P. 2004. PCR amplification‐based microbial typing. In Molecular Microbiology: Diagnostics Principles and Practice (D.H. Persing, F.C. Tenover, J. Versalovic, Y.‐W. Tang, E.R. Unger, D.A. Relman, and T.J. White, eds.) pp. 197‐221. ASM Press, Washington, DC.
   Ronaghi, M., Uhlén, M., and Nyrén, P. 1998. A sequencing method based on real‐time pyrophosphate. Science 281:363‐365.
   Rozen, S. and Skaletsky, H.J. 2000. Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols: Methods in Molecular Biology (S. Krawetz and S. Misener, eds.) pp. 365‐386. Humana Press, Totowa, NJ.
   Schaade, L., Kockelkorn, P., Ritter, K., and Kleines, M. 2001. Detection of cytomegalovirus DNA in human specimens by LightCycler PCR: Melting point analysis is mandatory to detect virus strains with point mutations in the target sequence of the hybridization probes. J. Clin. Microbiol. 39:3809.
   Versalovic, J. 2003. Is real‐time detection of drug‐resistant Staphylococcus aureus worth considering? Arch. Pathol. Lab. Med. 127:784‐785.
   Versalovic, J., Koeuth, T., and Lupski, J.R. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucl. Acids Res. 19:6823‐6831.
   Wittwer, C.T. and Kusukawa, N. 2004. Real‐time PCR. In Molecular Microbiology: Diagnostics Principles and Practice (D.H. Persing, F.C. Tenover, J. Versalovic, Y.‐W. Tang, E.R. Unger, D.A. Relman, and T.J. White, eds.) pp. 71‐84. ASM Press, Washington, DC.
Key References
   Hill and Caliendo, 2004. See above.
  A brief overview of viral load testing for not only HIV‐1, but also hepatitis B and C viruses, cytomegalovirus, and Epstein‐Barr virus using quantitative PCR.
   Mackay, 2004. See above.
  Current review of real‐time PCR and its applications.
   Wittwer and Kusukawa, 2004. See above.
  Discussion of technical aspects of real‐time PCR, aided by numerous tables and graphs.
   Jonasson et al., 2002. See above.
  First description of 16S rDNA pyrosequencing.
   Healy et al., 2005. See above.
  Definitive work on microbial typing with rep‐PCR by its inventors.
Internet Resources
   http://www.amptestdirectory.org
  Infectious diseases test directory of the Association for Molecular Pathology.
   http://www.roche‐applied‐science.com/lightcycler‐online
  Useful information from Roche Diagnostics for the LightCycler platform.
   http://www.biotagebio.com
  Pyrosequencing site of Biotage.
   http://rdp.cme.msu.edu/index.jsp
  Ribosomal Database Project site of the Center for Microbial Ecology of Michigan State University.
   http://www.bacbarcodes.com
  Information on the DiversiLab system from Bacterial Barcodes.
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