Skip to main content
NCBI home page
Journal of Clinical Pathology logoLink to Journal of Clinical Pathology
. 1998 Aug;51(8):617–620. doi: 10.1136/jcp.51.8.617

Detection of Aspergillus fumigatus PCR products by a microtitre plate based DNA hybridisation assay.

H A Fletcher 1, R C Barton 1, P E Verweij 1, E G Evans 1
PMCID: PMC500857  PMID: 9828823

Abstract

AIMS: To develop a DNA based plate hybridisation assay for the detection of polymerase chain reaction (PCR) products amplified from Aspergillus fumigatus DNA; and to determine the sensitivity of this technique and compare it with Southern blotting. METHODS: A half-log dilution series of DNA extracted from A fumigatus was amplified with specific primers, one of which was 5' end labelled with biotin. PCR products were subsequently detected by agarose gel electrophoresis, Southern blotting, and binding of the products to a streptavidin coated microtitre well, followed by non-radioactive colorimetric detection. Amplification was carried out 10 times for each DNA dilution and a plot of initial DNA concentration against signal intensity was made. RESULTS: A DNA concentration of 1.5 pg could be detected by agarose gel electrophoresis and Southern blotting with a non-radioactively labelled aspergillus specific probe; 1.5 pg was detectable by streptavidin binding of the PCR products to a microtitre plate. The signal from the microtitre plate detection was proportional to the amount of DNA in the PCR reaction on a log-log scale between 100 and 1 pg of DNA. CONCLUSIONS: A DNA based plate hybridisation assay for the detection of A fumigatus PCR products is as sensitive as Southern blotting. However, results are obtained in three hours rather than the three days required for agarose gel electrophoresis, blotting, hybridisation, and detection.

Full text

PDF
617

Images in this article

618Image
on p.618

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bretagne S., Costa J. M., Marmorat-Khuong A., Poron F., Cordonnier C., Vidaud M., Fleury-Feith J. Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. J Clin Microbiol. 1995 May;33(5):1164–1168. doi: 10.1128/jcm.33.5.1164-1168.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Einsele H., Hebart H., Roller G., Löffler J., Rothenhofer I., Müller C. A., Bowden R. A., van Burik J., Engelhard D., Kanz L. Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol. 1997 Jun;35(6):1353–1360. doi: 10.1128/jcm.35.6.1353-1360.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Haynes K. A., Westerneng T. J., Fell J. W., Moens W. Rapid detection and identification of pathogenic fungi by polymerase chain reaction amplification of large subunit ribosomal DNA. J Med Vet Mycol. 1995 Sep-Oct;33(5):319–325. doi: 10.1080/02681219580000641. [DOI] [PubMed] [Google Scholar]
  4. Hopfer R. L., Walden P., Setterquist S., Highsmith W. E. Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis. J Med Vet Mycol. 1993;31(1):65–75. doi: 10.1080/02681219380000071. [DOI] [PubMed] [Google Scholar]
  5. Hopwood V., Johnson E. M., Cornish J. M., Foot A. B., Evans E. G., Warnock D. W. Use of the Pastorex aspergillus antigen latex agglutination test for the diagnosis of invasive aspergillosis. J Clin Pathol. 1995 Mar;48(3):210–213. doi: 10.1136/jcp.48.3.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Melchers W. J., Verweij P. E., van den Hurk P., van Belkum A., De Pauw B. E., Hoogkamp-Korstanje J. A., Meis J. F. General primer-mediated PCR for detection of Aspergillus species. J Clin Microbiol. 1994 Jul;32(7):1710–1717. doi: 10.1128/jcm.32.7.1710-1717.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mitchell T. G., Sandin R. L., Bowman B. H., Meyer W., Merz W. G. Molecular mycology: DNA probes and applications of PCR technology. J Med Vet Mycol. 1994;32 (Suppl 1):351–366. doi: 10.1080/02681219480000961. [DOI] [PubMed] [Google Scholar]
  8. Mullins J., Seaton A. Fungal spores in lung and sputum. Clin Allergy. 1978 Sep;8(5):525–533. doi: 10.1111/j.1365-2222.1978.tb01506.x. [DOI] [PubMed] [Google Scholar]
  9. Reddy L. V., Kumar A., Kurup V. P. Specific amplification of Aspergillus fumigatus DNA by polymerase chain reaction. Mol Cell Probes. 1993 Apr;7(2):121–126. doi: 10.1006/mcpr.1993.1016. [DOI] [PubMed] [Google Scholar]
  10. Roymans R. T., Onland G., Postma B. H. One-day detection of PCR amplified Chlamydia trachomatis DNA in clinical samples: ELISA versus Southern blot hybridisation. J Clin Pathol. 1996 Jul;49(7):581–583. doi: 10.1136/jcp.49.7.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Sandhu G. S., Kline B. C., Stockman L., Roberts G. D. Molecular probes for diagnosis of fungal infections. J Clin Microbiol. 1995 Nov;33(11):2913–2919. doi: 10.1128/jcm.33.11.2913-2919.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Shin J. H., Nolte F. S., Morrison C. J. Rapid identification of Candida species in blood cultures by a clinically useful PCR method. J Clin Microbiol. 1997 Jun;35(6):1454–1459. doi: 10.1128/jcm.35.6.1454-1459.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Spreadbury C., Holden D., Aufauvre-Brown A., Bainbridge B., Cohen J. Detection of Aspergillus fumigatus by polymerase chain reaction. J Clin Microbiol. 1993 Mar;31(3):615–621. doi: 10.1128/jcm.31.3.615-621.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Tang C. M., Holden D. W., Aufauvre-Brown A., Cohen J. The detection of Aspergillus spp. by the polymerase chain reaction and its evaluation in bronchoalveolar lavage fluid. Am Rev Respir Dis. 1993 Nov;148(5):1313–1317. doi: 10.1164/ajrccm/148.5.1313. [DOI] [PubMed] [Google Scholar]
  15. Verweij P. E., Stynen D., Rijs A. J., de Pauw B. E., Hoogkamp-Korstanje J. A., Meis J. F. Sandwich enzyme-linked immunosorbent assay compared with Pastorex latex agglutination test for diagnosing invasive aspergillosis in immunocompromised patients. J Clin Microbiol. 1995 Jul;33(7):1912–1914. doi: 10.1128/jcm.33.7.1912-1914.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Pathology are provided here courtesy of BMJ Publishing Group

RESOURCES