Detection of fungal spores from contaminated surfacesby the polymerase chain reaction

Alicia Herna ndez, Jose L. Martõ nez and Rafael P. Mellado*Centro Nacional de BiotecnologõÂa (CSIC), Campus de la Universidad AutoÂnoma, Cantoblanco, 28049 Madrid, Spain*Author for correspondence: Tel.: 34-1-5854547, Fax: 34-1-5854503, E-mail: rpmellado@samba.cnb.uam.es

Received in revised form 24 August 1998; accepted 4 September 1998

Keywords: Fungal spores, molecular detection, PCR

Summary

A method has been developed to detect fungal spores in dust samples collected from internal surfaces of air-conditioning ducts. The method is based on the utilization of the polymerase chain reaction (PCR) with speci®cprimers for fungal species. PCR ampli®cation is carried out directly in boiled samples avoiding time-consumingDNA preparation steps. The presence of bovine serum albumin in the reaction mixture overcame the inhibitorye�ect of the humic acids present in the dust.

Introduction

Indoor airborne microorganisms are a source of healthhazards with special concern in the transmission ofcommunity acquired and nosocomial infections (Gun-dermann 1980; Proctor 1980). Bacteria commonlypresent in indoor air include important pathogens suchas Staphylococcus, Streptococcus, Pseudomonas, Flavo-bacterium Legionella pneumophila or Mycobacteriumtuberculosis. Fungal pathogens are not very commonin indoor air; however the presence of non-pathogenicfungi and their products often causes hypersensitivereactions that could produce severe problems in allergicpersons (Smith et al. 1992; Su et al. 1992). The presenceof microorganisms (or spores of them) on the internalsurfaces of air-conditioning ducts is also a factor in the``sick building syndrome'' (Hodgson 1992). Moreover,fungal colonization of acoustic and thermal ®breglassinsulation materials used in air-conditioning systemsproduces several harmful volatile organic compoundsthat mght contribute to some of the health problemsassociated with sick building syndrome (Ezeonu et al.1994). The development of methods to detect microor-ganisms present in air-conditioning systems is ofimportance in studies related to the emergence andtransmission of indoor airborne-linked diseases. Tradi-tional monitoring of microorganisms from indoor airrelies on culture-based techniques. These techniques aretime-consuming and present several limitations becausethe culture is always biased against slowly growingmicroorganisms; some species could have nonculturablestates, being undetectable by common laboratory tests;

sampling may cause stress and damage of airbornemicroorganisms; and microorganisms present in highconcentrations can obscure the presence of less abund-ant ones, particularly if the former produce antibacterialagents. Detection of fungal spores is of particularconcern, because they may grow poorly and may requirecomplex and speci®c media for their germination andgrowth. Therefore the use of molecular techniques andparticularly DNA ampli®cation by the polymerase chainreaction (PCR), is of great help in monitoring thequality of indoor air (MacNeil et al. 1995); but whenapplied to fungi, DNA ampli®cation by PCR (Gibbs1990; Mullis et al. 1986) always implied lengthy proce-dures of DNA extraction from fungal mycelia. In thispaper we describe a simpli®ed method that allowsdetection of fungal spores in dust samples by PCR toamplify the DNA directly released from the sporeswithout the need to germinate and culture them.

Materials and Methods

Culture conditions

E. coli DH5a cells were grown in Luria-Bertani (LB)broth (Atlas 1993) with orbital shaking at 37 °C untilthe optical density at 600 nm was 1.5. The number ofviable cells present in the culture was determined byplating serial dilutions on LB agar plates. Penicilliumchrysogenum was grown in Potato Dextrose Agar(PDA) Petri dishes (Atlas 1993) at 30 °C, and thespores were obtained by ®ltering fungal mycelia through

World Journal of Microbiology & Biotechnology 15: 33±36, 1999. 33Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.

a cotton plug. The number of germinating spores wasdetermined by plating serial dilutions on PDA plates.Penicillium chrysogenum cells for DNA ampli®cationwere harvested from submerged cultures grown in LBmedium and the number of viable cells determined onLB agar plates.

PCR ampli®cation

General DNA manipulation procedures were as de-scribed by Sambrook et al. (1989). The primers B2F:5¢-ACTTTCGATGGTAGGATAG-3¢ and B4R: 5¢-TGATCGTCTTCGATCCCCTA-3¢ were used for thePCR reaction. Each PCR assay contained 10 ll of 10 ´Buffer A (166 mM (NH4)2SO4; 670 mM Tris-HCl pH8.8; 0.1% Tween-20), 1 mM MgCl2, 1 mM dNTPs,100 mM BSA, 40 pmol of each primer, 5 ll of the boiledsample and 2.5 u of Taq DNA polymerase (Ecogen,S.R.L) in a ®nal volume of 100 ll. Ampli®cationreactions were carried out on a thermocycler (PTC-100HJ Research, Inc) for 30 cycles including 95 °C for1 min 45 °C for 1 min and 72 °C for 2 min. Reactionwas ended by a 10 min additional incubation step at72 °C. The ampli®ed products were analysed by elec-

trophoresis on 1% agarose gels and the DNA bandsvisualized by ethidium bromide staining.

Results and Discussion

To check whether fungal spores were detectable in dustsamples, the dust accumulated in a 10 cm2 area from theinternal surface of an air-conditioning duct was removedwith a cotton swab. The dust was resuspended in 10 mlof sterile water and dispensed in 1 ml aliquots. Di�erentamounts of Penicillium chrysogenum germinating spores,ranging from none to 10000, and the same range ofP. chrysogenum viable cells, were added to the tubes andthe resulting mixtures were incubated at 100 °C for tenminutes. After boiling, the mixtures were centrifuged forten minutes at 13000 rev/min in a Eppendorf minifuge toremove particulate matter and 5 ll of each supernatantwere directly used for PCR ampli®cation. The primersB2F: 5¢-ACTTTCGATGGTAGGATAG-3¢ and B4R:5¢-TGATCGTCTTCGATCCCCTA-3¢ were used forthe PCR reaction. Both primers derive from conservedsequences of 18S rDNA shared by most fungi (Ma-kimura et al. 1994).

Figure 1. Ampli®cation of fungal DNA from fun-

gal contaminated dust. (A) PCR ampli®cation of

DNA extracted from fungal spores. Lane 1, DNA

fragments ranging from 72 bp to 4300 bp resulting

from restriction of bacteriophage F29 DNA with

the endonuclease HindIII carried as molecular size

markers; lane 2, dust; lane 3, sterile water; lanes 4

to 8, dust containing 1, 10, 100, 1000 and 10000

spores, respectively; lane 9, 10000 spores not mixed

with dust. (B) PCR ampli®cation of DNA extracted

from fungal cells. Lane 1, molecular weight mark-

ers; lane 2, dust; lane 3, sterile water; lanes 4 to 8, 1,

10, 100, 1000 and 10000 cells, respectively, lane 9,

10000 cells not mixed with dust. The size of the

ampli®ed DNA fragment is indicated.

34 A. HernaÂndez et al.

The results of DNA ampli®cation are shown inFigure 1. The 687 bp long expected DNA fragment(Makimura et al. 1994) was clearly visible when thespores present in the reaction amounted to the equiv-alent 100 to 10000 colony forming units (Fig. 1A); theexpected DNA fragment was again visible when theequivalent amount of cells was present in the reactionmixture (Fig. 1B). A good correlation was observedbetween the amount of ampli®ed DNA and the esti-mated number of spores or cells, respectively. When 5 llof each of the DNA ampli®cation reactions was used asa template for a second PCR ampli®cation to furtherincrease the sensitivity of the method, as little as 10fungal spores were detected (not shown).When the same experiment was carried out with

equivalent amounts of E. coli DH5a viable cells, usingthe same set of fungal speci®c primers, no ampli®edDNA fragment was observed, as expected (Fig. 2).Direct boiling of the microorganism cells as a source

of template DNA for PCR ampli®cation has beensuccessfully used in di�erent microorganisms (Madicoet al. 1995). However, to our knowledge this methodhas never been used to obtain DNA from spores,probably because spores are highly resistant structures.The results obtained indicate that DNA from fungalspores suitable for PCR ampli®cation can also beobtained by direct boiling, avoiding lengthy DNAextraction methods. Puri®cation of nucleic acids frommicroorganisms contained in soil or in dust depositedin surfaces is not simple (Picard et al. 1992; Tsai &Olson 1992); the recovery is usually low, and thepresence of humic acids and phenolic compounds can

affect hybridization speci®city between primers andtemplate as well as the DNA polymerase polymeraseactivity (Tsai & Olson 1992; Tebbe & Vahjen 1993).The presence of high concentrations of BSA helped inalleviating the inhibitory effect of soil and dust con-taminants on the PCR ampli®cation (Fig. 1). Themethod is speci®c for the presence of fungal sporesnot only in air-conditioning systems but in differentenvironments, particularly in hospitals, where thepresence of fungal pathogens (Pfaller & Wenzel 1992)could be of major concern.

Acknowledgements

This work has been supported by Grants BIO94-072and BIO97-0650-C02-01 from the Spanish CICYT andby a Grant from Taj Europe S.L.

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