This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Loeffler, J.
Right arrow Articles by Einsele, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Loeffler, J.
Right arrow Articles by Einsele, H.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, February 2000, p. 586-590, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Quantification of Fungal DNA by Using Fluorescence Resonance Energy Transfer and the Light Cycler System

Juergen Loeffler,1,* Norbert Henke,1 Holger Hebart,1 Diethard Schmidt,1 Lars Hagmeyer,1 Ulrike Schumacher,2 and Hermann Einsele1

Medizinische Klinik, Abteilung II,1 and Hygieneinstitut,2 Abteilung Medizinische Mikrobiologie, Eberhard-Karls-Universität, 72076 Tübingen, Germany

Received 6 August 1999/Returned for modification 14 October 1999/Accepted 10 November 1999

The Light Cycler technique combines rapid in vitro amplification of DNA in glass capillaries with real-time species determination and quantification of DNA load. We have established a quantitative PCR protocol for two clinically important pathogens, Candida albicans and Aspergillus fumigatus. The sensitivity of the assay was comparable to those of previously described PCR protocols (5 CFU/ml). Specific detection of C. albicans and A. fumigatus could be achieved. The assay showed a high reproducibility of 96 to 99%. The assay was linear in a range between 101 and 104 Aspergillus conidia. As capillaries do not have to be reopened for post-PCR analysis, the risk of carryover contaminations could be minimized. The Light Cycler allowed quantification of the fungal loads in a limited number of clinical specimens from patients with hematological malignancies and histologically proven invasive fungal infections. Five of nine positive samples had fungal loads between 5 and 10 CFU/ml of blood, two of nine positive samples had fungal loads between 10 and 100 CFU/ml of blood, and two of nine samples had fungal loads of more than 100 CFU/ml of blood. All samples were also found to be PCR positive by PCR-enzyme-linked immunosorbent assay analysis.

* Corresponding author. Mailing address: Medizinische Klinik, Abt. II, Labor Prof. Dr. med. H. Einsele, Otfried-Mueller-Str. 10, 72076 Tuebingen, Germany. Phone: 49 7071 2987355. Fax: 49 7071 293179. E-mail: juergen.loeffler{at}med.uni-tuebingen.de.

Journal of Clinical Microbiology, February 2000, p. 586-590, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Soeta, N., Terashima, M., Gotoh, M., Mori, S., Nishiyama, K., Ishioka, K., Kaneko, H., Suzutani, T. (2009). An improved rapid quantitative detection and identification method for a wide range of fungi. J Med Microbiol 58: 1037-1044 [Abstract] [Full Text]  
  • Herrera, M. L., Vallor, A. C., Gelfond, J. A., Patterson, T. F., Wickes, B. L. (2009). Strain-Dependent Variation in 18S Ribosomal DNA Copy Numbers in Aspergillus fumigatus. J. Clin. Microbiol. 47: 1325-1332 [Abstract] [Full Text]  
  • Landlinger, C., Preuner, S., Willinger, B., Haberpursch, B., Racil, Z., Mayer, J., Lion, T. (2009). Species-Specific Identification of a Wide Range of Clinically Relevant Fungal Pathogens by Use of Luminex xMAP Technology. J. Clin. Microbiol. 47: 1063-1073 [Abstract] [Full Text]  
  • Coste, A., Lagane, C., Filipe, C., Authier, H., Gales, A., Bernad, J., Douin-Echinard, V., Lepert, J.-C., Balard, P., Linas, M.-D., Arnal, J.-F., Auwerx, J., Pipy, B. (2008). IL-13 Attenuates Gastrointestinal Candidiasis in Normal and Immunodeficient RAG-2-/- Mice via Peroxisome Proliferator-Activated Receptor-{gamma} Activation. J. Immunol. 180: 4939-4947 [Abstract] [Full Text]  
  • Metwally, L., Fairley, D. J., Coyle, P. V., Hay, R. J., Hedderwick, S., McCloskey, B., O'Neill, H. J., Webb, C. H., Elbaz, W., McMullan, R. (2008). Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR. J Med Microbiol 57: 296-303 [Abstract] [Full Text]  
  • Spiess, B., Seifarth, W., Hummel, M., Frank, O., Fabarius, A., Zheng, C., Morz, H., Hehlmann, R., Buchheidt, D. (2007). DNA Microarray-Based Detection and Identification of Fungal Pathogens in Clinical Samples from Neutropenic Patients. J. Clin. Microbiol. 45: 3743-3753 [Abstract] [Full Text]  
  • Press, R. D., Galderisi, C., Yang, R., Rempfer, C., Willis, S. G., Mauro, M. J., Druker, B. J., Deininger, M. W.N. (2007). A Half-Log Increase in BCR-ABL RNA Predicts a Higher Risk of Relapse in Patients with Chronic Myeloid Leukemia with an Imatinib-Induced Complete Cytogenetic Response. Clin. Cancer Res. 13: 6136-6143 [Abstract] [Full Text]  
  • Baskova, L., Landlinger, C., Preuner, S., Lion, T. (2007). The Pan-AC assay: a single-reaction real-time PCR test for quantitative detection of a broad range of Aspergillus and Candida species. J Med Microbiol 56: 1167-1173 [Abstract] [Full Text]  
  • Schabereiter-Gurtner, C., Selitsch, B., Rotter, M. L., Hirschl, A. M., Willinger, B. (2007). Development of Novel Real-Time PCR Assays for Detection and Differentiation of Eleven Medically Important Aspergillus and Candida Species in Clinical Specimens. J. Clin. Microbiol. 45: 906-914 [Abstract] [Full Text]  
  • Huang, A., Li, J.-W., Shen, Z.-Q., Wang, X.-W., Jin, M. (2006). High-throughput identification of clinical pathogenic fungi by hybridization to an oligonucleotide microarray.. J. Clin. Microbiol. 44: 3299-3305 [Abstract] [Full Text]  
  • White, P. L., Barton, R., Guiver, M., Linton, C. J., Wilson, S., Smith, M., Gomez, B. L., Carr, M. J., Kimmitt, P. T., Seaton, S., Rajakumar, K., Holyoake, T., Kibbler, C. C., Johnson, E., Hobson, R. P., Jones, B., Barnes, R. A., on behalf of the United Kingdom Fungal Polymerase, (2006). A Consensus on Fungal Polymerase Chain Reaction Diagnosis?: A United Kingdom-Ireland Evaluation of Polymerase Chain Reaction Methods for Detection of Systemic Fungal Infections. J. Mol. Diagn. 8: 376-384 [Abstract] [Full Text]  
  • Espy, M. J., Uhl, J. R., Sloan, L. M., Buckwalter, S. P., Jones, M. F., Vetter, E. A., Yao, J. D. C., Wengenack, N. L., Rosenblatt, J. E., Cockerill, F. R. III, Smith, T. F. (2006). Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin. Microbiol. Rev. 19: 165-256 [Abstract] [Full Text]  
  • Scotter, J. M., Chambers, S. T. (2005). Comparison of Galactomannan Detection, PCR-Enzyme-Linked Immunosorbent Assay, and Real-Time PCR for Diagnosis of Invasive Aspergillosis in a Neutropenic Rat Model and Effect of Caspofungin Acetate. CVI 12: 1322-1327 [Abstract] [Full Text]  
  • Millon, L., Piarroux, R., Deconinck, E., Bulabois, C.-E., Grenouillet, F., Rohrlich, P., Costa, J.-M., Bretagne, S. (2005). Use of Real-Time PCR To Process the First Galactomannan-Positive Serum Sample in Diagnosing Invasive Aspergillosis. J. Clin. Microbiol. 43: 5097-5101 [Abstract] [Full Text]  
  • Halliday, C., Wu, Q. X., James, G., Sorrell, T. (2005). Development of a Nested Qualitative Real-Time PCR Assay To Detect Aspergillus Species DNA in Clinical Specimens. J. Clin. Microbiol. 43: 5366-5368 [Abstract] [Full Text]  
  • Bu, R., Sathiapalan, R. K, Ibrahim, M. M, Al-Mohsen, I., Almodavar, E., Gutierrez, M. I, Bhatia, K. (2005). Monochrome LightCycler PCR assay for detection and quantification of five common species of Candida and Aspergillus. J Med Microbiol 54: 243-248 [Abstract] [Full Text]  
  • Balashov, S. V., Gardiner, R., Park, S., Perlin, D. S. (2005). Rapid, High-Throughput, Multiplex, Real-Time PCR for Identification of Mutations in the cyp51A Gene of Aspergillus fumigatus That Confer Resistance to Itraconazole. J. Clin. Microbiol. 43: 214-222 [Abstract] [Full Text]  
  • O'Mahony, J., Hill, C. (2004). Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp. paratuberculosis DNA in Artificially Contaminated Milk. Appl. Environ. Microbiol. 70: 4561-4568 [Abstract] [Full Text]  
  • de Aguirre, L., Hurst, S. F., Choi, J. S., Shin, J. H., Hinrikson, H. P., Morrison, C. J. (2004). Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay. J. Clin. Microbiol. 42: 3495-3504 [Abstract] [Full Text]  
  • Maaroufi, Y., Ahariz, N., Husson, M., Crokaert, F. (2004). Comparison of Different Methods of Isolation of DNA of Commonly Encountered Candida Species and Its Quantitation by Using a Real-Time PCR-Based Assay. J. Clin. Microbiol. 42: 3159-3163 [Abstract] [Full Text]  
  • Kawazu, M., Kanda, Y., Nannya, Y., Aoki, K., Kurokawa, M., Chiba, S., Motokura, T., Hirai, H., Ogawa, S. (2004). Prospective Comparison of the Diagnostic Potential of Real-Time PCR, Double-Sandwich Enzyme-Linked Immunosorbent Assay for Galactomannan, and a (1->3)-{beta}-D-Glucan Test in Weekly Screening for Invasive Aspergillosis in Patients with Hematological Disorders. J. Clin. Microbiol. 42: 2733-2741 [Abstract] [Full Text]  
  • Frade, J. P., Warnock, D. W., Arthington-Skaggs, B. A. (2004). Rapid Quantification of Drug Resistance Gene Expression in Candida albicans by Reverse Transcriptase LightCycler PCR and Fluorescent Probe Hybridization. J. Clin. Microbiol. 42: 2085-2093 [Abstract] [Full Text]  
  • Maaroufi, Y., De Bruyne, J.-M., Duchateau, V., Georgala, A., Crokaert, F. (2004). Early Detection and Identification of Commonly Encountered Candida Species from Simulated Blood Cultures by Using a Real-Time PCR-Based Assay. J. Mol. Diagn. 6: 108-114 [Abstract] [Full Text]  
  • Hong, K. M., Najjar, H., Hawley, M., Press, R. D. (2004). Quantitative Real-Time PCR with Automated Sample Preparation for Diagnosis and Monitoring of Cytomegalovirus Infection in Bone Marrow Transplant Patients. Clin. Chem. 50: 846-856 [Abstract] [Full Text]  
  • Johnson, M. D., MacDougall, C., Ostrosky-Zeichner, L., Perfect, J. R., Rex, J. H. (2004). Combination Antifungal Therapy. Antimicrob. Agents Chemother. 48: 693-715 [Full Text]  
  • Challier, S., Boyer, S., Abachin, E., Berche, P. (2004). Development of a Serum-Based Taqman Real-Time PCR Assay for Diagnosis of Invasive Aspergillosis. J. Clin. Microbiol. 42: 844-846 [Abstract] [Full Text]  
  • Sendid, B., Jouault, T., Coudriau, R., Camus, D., Odds, F., Tabouret, M., Poulain, D. (2004). Increased Sensitivity of Mannanemia Detection Tests by Joint Detection of {alpha}- and {beta}-Linked Oligomannosides during Experimental and Human Systemic Candidiasis. J. Clin. Microbiol. 42: 164-171 [Abstract] [Full Text]  
  • Hsu, M.-C., Chen, K.-W., Lo, H.-J., Chen, Y.-C., Liao, M.-H., Lin, Y.-H., Li, S.-Y. (2003). Species identification of medically important fungi by use of real-time LightCycler PCR. J Med Microbiol 52: 1071-1076 [Abstract] [Full Text]  
  • Rantakokko-Jalava, K., Laaksonen, S., Issakainen, J., Vauras, J., Nikoskelainen, J., Viljanen, M. K., Salonen, J. (2003). Semiquantitative Detection by Real-Time PCR of Aspergillus fumigatus in Bronchoalveolar Lavage Fluids and Tissue Biopsy Specimens from Patients with Invasive Aspergillosis. J. Clin. Microbiol. 41: 4304-4311 [Abstract] [Full Text]  
  • Sanguinetti, M., Posteraro, B., Pagano, L., Pagliari, G., Fianchi, L., Mele, L., La Sorda, M., Franco, A., Fadda, G. (2003). Comparison of Real-Time PCR, Conventional PCR, and Galactomannan Antigen Detection by Enzyme-Linked Immunosorbent Assay Using Bronchoalveolar Lavage Fluid Samples from Hematology Patients for Diagnosis of Invasive Pulmonary Aspergillosis. J. Clin. Microbiol. 41: 3922-3925 [Abstract] [Full Text]  
  • Maaroufi, Y., Heymans, C., De Bruyne, J.-M., Duchateau, V., Rodriguez-Villalobos, H., Aoun, M., Crokaert, F. (2003). Rapid Detection of Candida albicans in Clinical Blood Samples by Using a TaqMan-Based PCR Assay. J. Clin. Microbiol. 41: 3293-3298 [Abstract] [Full Text]  
  • Spiess, B., Buchheidt, D., Baust, C., Skladny, H., Seifarth, W., Zeilfelder, U., Leib-Mosch, C., Morz, H., Hehlmann, R. (2003). Development of a LightCycler PCR Assay for Detection and Quantification of Aspergillus fumigatus DNA in Clinical Samples from Neutropenic Patients. J. Clin. Microbiol. 41: 1811-1818 [Abstract] [Full Text]  
  • Holzl, G., Stocher, M., Leb, V., Stekel, H., 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 [Abstract] [Full Text]  
  • White, P. L., Shetty, A., Barnes, R. A. (2003). Detection of seven Candida species using the Light-Cycler system. J Med Microbiol 52: 229-238 [Abstract] [Full Text]  
  • Li, W., Tanaka, K., Chiba, Y., Kimura, T., Morioka, K., Uesaka, T., Ihaya, A., Sasaki, M., Tsuda, T., Yamada, N. (2003). Role of MMPs and plasminogen activators in angiogenesis after transmyocardial laser revascularization in dogs. Am. J. Physiol. Heart Circ. Physiol. 284: H23-H30 [Abstract] [Full Text]  
  • Taylor, D. E., Rooker, M., Keelan, M., Ng, L.-K., Martin, I., Perna, N. T., Burland, N. T. V., Blattner, F. R. (2002). Genomic Variability of O Islands Encoding Tellurite Resistance in Enterohemorrhagic Escherichia coli O157:H7 Isolates. J. Bacteriol. 184: 4690-4698 [Abstract] [Full Text]  
  • Luo, G., Mitchell, T. G. (2002). Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR. J. Clin. Microbiol. 40: 2860-2865 [Abstract] [Full Text]  
  • Yeo, S. F., Wong, B. (2002). Current Status of Nonculture Methods for Diagnosis of Invasive Fungal Infections. Clin. Microbiol. Rev. 15: 465-484 [Abstract] [Full Text]  
  • Costa, C., Costa, J.-M., Desterke, C., Botterel, F., Cordonnier, C., Bretagne, S. (2002). Real-Time PCR Coupled with Automated DNA Extraction and Detection of Galactomannan Antigen in Serum by Enzyme-Linked Immunosorbent Assay for Diagnosis of Invasive Aspergillosis. J. Clin. Microbiol. 40: 2224-2227 [Abstract] [Full Text]  
  • Loeffler, J., Schmidt, K., Hebart, H., Schumacher, U., 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 [Abstract] [Full Text]  
  • McILHATTON, B. P., KEATING, C., CURRAN, M. D., McMULLIN, M.-F., BARR, J. G., MADRIGAL, J. A., MIDDLETON, D. (2002). Identification of medically important pathogenic fungi by reference strand-mediated conformational analysis (RSCA). J Med Microbiol 51: 468-478 [Abstract] [Full Text]  
  • Bialek, R., Weiss, M., Bekure-Nemariam, K., Najvar, L. K., Alberdi, M. B., Graybill, J. R., Reischl, U. (2002). Detection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays. CVI 9: 461-469 [Abstract] [Full Text]  
  • Bowman, J. C., Abruzzo, G. K., Anderson, J. W., Flattery, A. M., Gill, C. J., Pikounis, V. B., Schmatz, D. M., Liberator, P. A., Douglas, C. M. (2001). Quantitative PCR Assay To Measure Aspergillus fumigatus Burden in a Murine Model of Disseminated Aspergillosis: Demonstration of Efficacy of Caspofungin Acetate. Antimicrob. Agents Chemother. 45: 3474-3481 [Abstract] [Full Text]  
  • Okeke, C. N., Tsuboi, R., Ogawa, H. (2001). Quantification of Candida albicans Actin mRNA by the LightCycler System as a Means of Assessing Viability in a Model of Cutaneous Candidiasis. J. Clin. Microbiol. 39: 3491-3494 [Abstract] [Full Text]  
  • Hein, I., Lehner, A., Rieck, P., Klein, K., Brandl, E., Wagner, M. (2001). Comparison of Different Approaches To Quantify Staphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese. Appl. Environ. Microbiol. 67: 3122-3126 [Abstract] [Full Text]  
  • Kessler, H. H., Muhlbauer, G., Stelzl, E., Daghofer, E., Santner, B. I., Marth, E. (2001). Fully Automated Nucleic Acid Extraction: MagNA Pure LC. Clin. Chem. 47: 1124-1126 [Full Text]  
  • Loeffler, J., Hebart, H., Cox, P., Flues, N., Schumacher, U., Einsele, H. (2001). Nucleic Acid Sequence-Based Amplification of Aspergillus RNA in Blood Samples. J. Clin. Microbiol. 39: 1626-1629 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»