Genus- and Species-Specific PCR Detection Methods

  • Jan Springer
  • Jürgen LöfflerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1508)


PCR-based detection of fungal pathogens offers a sensitive and specific tool for the diagnosis of invasive fungal infections. A large variety of different clinical specimen types can be used as original material. However, certain precautions, in addition to the published MIQE guidelines [1], need to be taken to prevent contaminations from airborne fungal spores and PCR reagents. In addition, the European Aspergillus PCR Initiative (EAPCRI) recently defined standards for Aspergillus PCR [2, 3], following these recommendations leads to superior sensitivity. The combination of fungal PCR with the galactomannan ELISA assay increases the sensitivity for the detection of Aspergillus DNA from blood, compared to a single assay only [4, 5].

Key words

DNA Detection Fungi PCR Amplification Broad-range PCR Species-specific method 


  1. 1.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622, doi:clinchem.2008.112797 [pii] 10.1373/clinchem.2008.112797CrossRefPubMedGoogle Scholar
  2. 2.
    White PL, Bretagne S, Klingspor L, Melchers WJ, McCulloch E, Schulz B, Finnstrom N, Mengoli C, Barnes RA, Donnelly JP, Loeffler J (2010) Aspergillus PCR: one step closer to standardization. J Clin Microbiol 48(4):1231–1240, doi:JCM.01767-09 [pii] 10.1128/JCM.01767-09Google Scholar
  3. 3.
    White PL, Mengoli C, Bretagne S, Cuenca-Estrella M, Finnstrom N, Klingspor L, Melchers WJ, McCulloch E, Barnes RA, Donnelly JP, Loeffler J (2011) Evaluation of Aspergillus PCR protocols for testing serum specimens. J Clin Microbiol 49(11):3842–3848, doi:JCM.05316-11 [pii] 10.1128/JCM.05316-11CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Rogers TR, Morton CO, Springer J, Conneally E, Heinz W, Kenny C, Frost S, Einsele H, Loeffler J (2013) Combined real-time PCR and galactomannan surveillance improves diagnosis of invasive aspergillosis in high risk patients with haematological malignancies. Br J Haematol. Accepted for PublicationGoogle Scholar
  5. 5.
    Morrissey CO, Chen SCA, Sorrell TC, Milliken S, Bardy PG, Bradstock KF, Szer J, Halliday CL, Gilroy NM, Moore J, Schwarer AP, Guy S, Bajel A, Tramontana AR, Spelman T, Slavin MA, Australasian Leukaemia Lymphoma Group, The Australia, New Zealand Mycology Interest Group (2013) Galactomannan and PCR versus culture and histology for directing use of antifungal treatment for invasive aspergillosis in high-risk haematology patients: a randomised controlled trial. Lancet Infect Dis 13(6):519–528CrossRefPubMedGoogle Scholar
  6. 6.
    Baddley JW, Andes DR, Marr KA, Kontoyiannis DP, Alexander BD, Kauffman CA, Oster RA, Anaissie EJ, Walsh TJ, Schuster MG, Wingard JR, Patterson TF, Ito JI, Williams OD, Chiller T, Pappas PG (2010) Factors associated with mortality in transplant patients with invasive aspergillosis. Clin Infect Dis 50(12):1559–1567CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Marr KA, Carter RA, Crippa F, Wald A, Corey L (2002) Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin Infect Dis 34(7):909–917CrossRefPubMedGoogle Scholar
  8. 8.
    Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr KA, Morrison VA, Segal BH, Steinbach WJ, Stevens DA, van Burik J-A, Wingard JR, Patterson TF, Infectious Diseases Society of America (2008) Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 46(3):327–360CrossRefPubMedGoogle Scholar
  9. 9.
    Luther K, Rohde M, Sturm K, Kotz A, Heesemann J, Ebel F (2008) Characterisation of the phagocytic uptake of Aspergillus fumigatus conidia by macrophages. Microbes Infect 10(2):175–184Google Scholar
  10. 10.
    Loeffler J, Mengoli C, Springer J, Bretagne S, Cuenca-Estrella M, Klingspor L, Lagrou K, Melchers WJ, Morton CO, Barnes RA, Donnelly JP, White PL (2015) Analytical comparison of in vitro-spiked human serum and plasma for PCR-based detection of Aspergillus fumigatus DNA: a study by the European Aspergillus PCR initiative. J Clin Microbiol 53(9):2838–2845, doi:JCM.00906-15 [pii] 10.1128/JCM.00906-15Google Scholar
  11. 11.
    Einsele H, Hebart H, Roller G, Loffler J, Rothenhofer I, Muller CA, Bowden RA, van Burik J, Engelhard D, Kanz L, Schumacher U (1997) Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 35(6):1353–1360PubMedPubMedCentralGoogle Scholar
  12. 12.
    Springer J, Loeffler J, Heinz W, Schlossnagel H, Lehmann M, Morton O, Rogers TR, Schmitt C, Frosch M, Einsele H, Kurzai O (2011) Pathogen-specific DNA enrichment does not increase sensitivity of PCR for diagnosis of invasive aspergillosis in neutropenic patients. J Clin Microbiol 49(4):1267–1273, doi: JCM.01679-10 [pii] 10.1128/JCM.01679-10CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Bialek R, Konrad F, Kern J, Aepinus C, Cecenas L, Gonzalez GM, Just-Nubling G, Willinger B, Presterl E, Lass-Florl C, Rickerts V (2005) PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue. J Clin Pathol 58(11):1180–1184CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Hrncirova K, Lengerova M, Kocmanova I, Racil Z, Volfova P, Palousova D, Moulis M, Weinbergerova B, Winterova J, Toskova M, Pospisilova S, Mayer J (2010) Rapid detection and identification of mucormycetes from culture and tissue samples by use of high-resolution melt analysis. J Clin Microbiol 48(9):3392–3394, doi: JCM.01109-10 [pii] 10.1128/JCM.01109-10CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Nucci F, Nouer SA, Capone D, Anaissie E, Nucci M (2015) Fusariosis. Semin Respir Crit Care Med 36(5):706–714CrossRefPubMedGoogle Scholar
  16. 16.
    Hue FX, Huerre M, Rouffault MA, de Bievre C (1999) Specific detection of Fusarium species in blood and tissues by a PCR technique. J Clin Microbiol 37(8):2434–2438Google Scholar
  17. 17.
    Babouee Flury B, Weisser M, Prince SS, Bubendorf L, Battegay M, Frei R, Goldenberger D (2014) Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? BMC Infect Dis 14:692CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Medizinische Klinik IIUniversitätklinikum WürzburgWürzburgGermany

Personalised recommendations