Campisi, Enza

Faggi, Elisabetta

Pini, Gabriella

English

Florence Research

Use of magnetic beads to extract fungal DNAE. Faggi, G. Pini and E. CampisiDepartment of Public Health, Microbiology Unit, University of Florence, Florence, ItalySummary Authors compare two methods of extracting DNA from different fungi: the classicmethod with phenol/chloroform (P/C) and that with magnetic beads. Both were testedon Candida albicans and Cryptococcus neoformans var. neoformans, belonging to the yeastgroup and Microsporum canis, M. gypseum, Trichophyton rubrum, T. interdigitale,T. ajelloi, Epidermophyton floccosum, belonging to the dermatophytes group. Extractionproducts underwent polymerase chain reaction (PCR) fingerprinting with theappropriate primers to point out any disagreement in the genomic profiles. Afterhaving determined that the genomic profiles obtained from the DNA extracted from thesame strain with the two methods correspond perfectly, the authors concluded that theextraction method with magnetic beads from fungal cells is simpler and quicker thanwith P/C extraction, greatly facilitating the obtainment of fungal DNA.Key words: DNA extraction, fungal DNA, magnetic beads.IntroductionRecent molecular methods for epidemiological sub-typing or taxonomic research, such as random amplifiedpolymorphic DNA (RAPD) or restriction fragmentlength polymorphism (RFLP) and polymerase chainreaction (PCR)-based diagnostic techniques, have deter-mined the need for a rapid and easy method ofextracting genomic DNA from different microbial agentsand from viruses. However, for fungi the presence of acomplex cell wall makes working times longer since anenzymatic and/or mechanical pretreatment which elim-inates this structure is necessary; nevertheless it hasbeen possible to extract fungal DNA from fungi bothnon-pathogenic and pathogenic in man, even if withdifferentiated methods and not always quick.In 1989, Morrissey et al.1 have introduced the use ofmagnetic beads to capture the target DNA hybridizedwith a probe; these magnetic beads were then success-fully used for extracting DNA from various tissues orinfectious agents simply and quickly2–8 allowing to savea noticeable amount of time in comparison withtraditional methods, and avoiding the manipulation ofharmful substances.Nevertheless, the first news of the use of this methodof extracting DNA from fungi is from Rudi et al.9 andLoeffler et al.10 who in 1997 applied it respectively tofruit bodies and mycelia of various fungi and on Candidaalbicans and Aspergillus niger, while Scott et al.11 in2000 used it for a fungus with agricultural interest,Claviceps africana.We used magnetic beads (Dynabeads DNA DirectSystem I; Dynal, Oslo, Norway), for extracting DNAfrom the yeasts C. albicans, and Cryptococcus neoformansvar. neoformans and some species of dermatophytes(Microsporum canis, M. gypseum, Trichophyton rubrum,T. interdigitale, T. ajelloi, Epidermophyton floccosum), andwe compared it with the classical extraction methodwith phenol/chloroform (P/C),12 in order to verify anactual greater speed in obtaining a detectable quantityof PCR-ready DNA.As for dermatophytes, the DNA was also extractedfrom 3-day cultures (young colonies, YC) and fromstrains without typical morphological characteristics(SWTMC), with the aim of speeding up the method as awhole, and from dead dermatophytes to verify thestability of the genomic characteristics after death.13Correspondence: Dr Enza Campisi, Department of Public Health, Micro-biology Unit, Viale G. B. Morgagni, 48, I-50134 Florence, Italy.Tel.: +39-55-3262444. Fax: +39-55-3262446.E-mail: ecampisi@mail.unifi.itAccepted for publication 18 July 2003Original article 2005 Blackwell Publishing Ltd • Mycoses, 48, 3–7 3The DNA extracted with the two methods underwenta PCR with the appropriate primers, in order to comparethe genomic profiles obtained and to verify identity.In fact, on the basis of our previous studies concern-ing fungi14, 15 we have established a strict correspon-dence between species of fungus and genomic profile.Materials and methodsCandida albicansDynabeads DNA extraction. We tested 40 strains isolatedfrom the oral cavity of HIV-positive patients and HIV-negative subjects, by means of the Dynal, partlymodified. The yeasts were grown on a minimummedium (MM) (yeast nitrogen base, agar 2%, glucose2%; pH 7.4) for 24 h at 35 C, then the fungal growthwas taken and washed with 5 ml of SCS (sorbitol1 mol l)1, trisodium citrate 20 mmol l)1; pH 5.8)centrifuged and resuspended in 200 ll of H2O (about109 yeast cells ml)1).About 20 ll of the suspension are incubated with200 ll of Dynabeads (paramagnetic polystyrene beadsin lysis buffer) for 10 min at 65 C, so as to obtain celllysis and the adsorption of the released DNA to theirsurface. This step is followed by magnetic separation ofthe intact DNA/Dynabeads complex and by subsequentwashing with washing buffer, which removes anyresidual contaminant and eliminates potential PCRinhibitors. The DNA/Dynabeads complex was thenresuspended in TE buffer [Tris-HCl 10 mmol l)1 (pH8), ethylenediaminetetraacetic acid (EDTA) 1 mmol l)1]and DNA eluted for 5 min at 65 C; then it was readyfor PCR or to be stored at )20 C.Phenol-chloroform DNA extraction. We examined eight(of the preceding 40 ones) strains. The yeasts weregrown on MM for 16 h at 35 C, then the fungalgrowth (about 109 cells ml)1) was taken and washedwith 5 ml of SCS and centrifuged at 3000 g per 7 min.The sediment was treated with 2 ml of lysing enzymefrom Trichoderma harzianum (Sigma, St Louis, MO, USA),at the final concentration of 5 mg ml)1 in SCS andincubated for 2 h at 35 C.After having determined by means of a countingchamber that 70% of the cells were protoplasts, it wascentrifuged at 3000 g for 5 min, the supernatant wasdiscarded and 2.5 ml of lysis solution was added(pH 8) [Tris-HCl 10 mmol l)1, EDTA 0.1 mol l)1,proteinase K 2 mg ml)1, N-laurosylsarcosine (Sigma)1%]. It was incubated at 35 C over night. Precipi-tation with isopropanol and extraction with P/Cfollowed, according to the method described by Varmaand Kwon-Chung.12PCR amplification. The PCR was performed with primerA2 (5¢-TGGTCGCGGC-3¢).16Cryptococcus neoformans var. neoformansDynabeads DNA extraction. We examined 25 strains.Because of the capsule presence, the procedure was thesame used for C. albicans in the P/C extraction untilprotoplasts were obtained. After centrifugation 100 llof distilled H2O were added to the sediment and with20 ll of suspension we proceeded as for DynabeadsDNA extraction of C. albicans.Phenol-chloroform DNA extraction. We examined 90strains from pathological specimens (Table 1).The yeast cells were previously deprived of thecapsule, then lysed in a solution of EDTA, Tris-HCl,N-laurosylsarcosine (Sigma) 1%, proteinase K. The DNAwas then extracted by means of P/C, as described forC. albicans.PCR amplification. The PCR was performed with primer(GACA)4.14DermatophytesDynabeads DNA extraction. It was performed onM. canis, M. gypseum, T. rubrum, T. interdigitale,T. ajelloi, E. floccosum for a total of 140 strains.15They were grown in Sabouraud glucose agar (SDA) at25 C; after 2 weeks a little mycelium was cut fromthe agar and transferred to Sabouraud glucose brothfor 2 more weeks at 25 C. The superficial mycelialgrowth was transferred to a mortar, washed withTable 1 Typology of the examined samples.MycetesNumberof strains Source OriginCandida albicans 40 Oral cavity HIV+ and HIV)Cryptococcus neoformans 90 CSF (58)1 HIV+Blood (15) HIV+Urine (8) HIV+Sputum (6) HIV+Glands (1) HIV+Skin (2) HIV+Dermatophytes 140 Man, dog, cat(see Ref. 13)1Number of samples/90.CSF, cerebrospinal fluid.E. Faggi et al.4  2005 Blackwell Publishing Ltd • Mycoses, 48, 3–7distilled water and pestled. About 20 ll of pestledmycelium was transferred to an Eppendorf tube andincubated with 200 ll of Dynabeads as previouslydescribed.Young colonies. This test was conducted on 12 ofpreceding strains belonging to the following species:M. canis, T. mentagrophytes, T. interdigitale, T. rubrum,M. gypseum, E. floccosum. The strains were grown inSDA (Difco, Detroit, MI, USA) at 25 C; after 3 days acolony (mean diameter 5 mm) was taken andtransferred to an Eppendorf tube containing 40 ll ofsterile distilled water. The mycelium was homogenizedwith a manual homogenizer (Micro-Grinder, PBIInternational, Milan, Italy) for 1 min. Then 400 ll ofDynabeads were added to the homogenized material andthe extraction was effected according to the previouslydescribed method.Strains without typical morphological characteristics. Westudied eight of preceding strains which had lost theirtypical morphological characteristics but which hadoriginally been identified as M. canis, T. rubrum,T. interdigitale. Some aerial mycelium was taken andwe proceeded to DNA extraction and PCR fingerprintingas described above.Dead strains. We studied 12 dead strains (DS) that hadoriginally been identified as M. canis, T. mentagrophytes,T. interdigitale, E. floccosum. By using a bacteriologicalspatula some mycelium was scraped out and DNA wasextracted as described above.Phenol-chloroform DNA extraction. This was performedon 10 strains (M. canis, T. interdigitale, T. rubrum) thathad been precultivated, as above. The surface myceliumwas collected, washed twice with SCS, and pestled. Weproceeded to extraction from all the pestled materialaccording to Bowman.17PCR amplification. The PCR was performed with primer(GACA)4.13, 15PCR amplificationEach DNA solution was diluted with TE buffer and itsUV-absorption spectrum was assayed using a BeckmanDU-64 spectrometer (Beckman Coulter, Fullerton, CA,USA). We estimated the concentration of nucleic acidsbased on the absorbance of 260 nm. In all themycetes, the yield of DNA with the two methodswas expressed as lg ml)1 and the degree of purity wascalculated as the ratio between optical density (OD)measured at 260 nm and that measured at 280 nm(OD 260/OD 280).The PCR was performed with primer (GACA)4. Eachsample of genomic DNA was amplified in duplicatein the same PCR and in repeated PCRs at differenttimes.Statistical analysisStatistical analysis to study variability between theextraction methods was carried out using the MicrosoftExcel 2000 statistical package, calculating mean, SDand by ANOVA.ResultsTable 1 shows the origin and the source of theexamined strains. Table 2 shows the strain samplesthe DNA was extracted from.The DNA of 237 strains was extracted by means ofDynabeads and that of 108 strains with the P/Cmethod. In all cases, the greatest number of strainstested with one method includes those tested with theother method, with the exception of the dermatophytesYC, SWTMC, DS for which only the Dynabeads test wasused.To verify the expediency of one method rather thananother, we compared the concentration and purity(arithmetical average of the values obtained for all thestrains tested in each group) of the DNA extracted bymeans of Dynabeads method with those obtainedthrough the classical extraction with P/C (Table 3).We observed that with the P/C method a definitelygreater concentration of DNA was obtained, 15 timesgreater for C. albicans, about nine times for Cr. neofor-mans and almost three times for the dermatophytes.Table 2 Samples of the fungi from which the DNA was extracted.MycetesNumber of tested strainsDynabeads Phenol/chloroformCandida albicans 40 8Cryptococcus neoformans 25 90Dermatophytes 140 10YC 12SWTMC 8DS 12Total 237 108YC, young colonies; SWTMC, strains without typical morphologi-cal characteristics; DS, dead strains.Magnetic beads for fungal DNA extraction 2005 Blackwell Publishing Ltd • Mycoses, 48, 3–7 5The purity grade was greater only for C. albicans,while for Cr. neoformans and the dermatophytes thevalue was about the same.Amplification through PCR with the appropriateprimers of the DNA extracted allowed us to comparethe quality of the genomic profiles obtained. Figure 1shows some representative examples of the profilesobtained from the strains tested. The profiles obtainedfrom the DNA extracted with the two methods wereperfectly identical: only the M. canis profile (lanes 9 and10) showed some more brightly coloured bands in theDNA extracted by P/C method.As the cell suspensions of compared strains at startwere different for the two extraction methods, weproceeded to compare the DNA mean yield, calculatingthe quantity of DNA extracted on average from everycell. As regard C. albicans there is no significantdifference about the yield (P ¼ 0.9), whereas as regardCr. neoformans the P/C method obtained a significantlyhigher yield (P < 0.001). As regard dermatophytes, wecannot know the number of cells present in the initialsuspension because these are mycelial hyphae, thereforethe yield was estimated not at cell but by the weight ofthe mycelia at start. Also in this case the yield by P/Cmethod was higher (P ¼ 0.0046).DiscussionFrom the data presented we draw the followingconclusions:The P/C method presents more advantages incomparison with Dynabeads under two aspects: thequantitative/qualitative one, because the rate yield/purity is higher and the economic one, because cost/test is definitely lower in comparison with magneticbeads.On the contrary, Dynabeads test is advantageous invarious aspects. The first one to mention is the easeexecution (any operator is able to execute easily thescheduled passages), moreover DNA is obtained inmuch briefer time (30–40 min vs. 24–25 h with P/Cextraction) and without manipulation of harmful sub-stances. There is no need for special laboratory equip-ment (extractor hood for chemical, supercentrifuge,etc.), but it is enough only the provided kit and amicropipette.In addition, we must remember the fate of the use ofthe extracted DNA.The DNA of the fungal strains tested by us wasextracted to undergo PCR fingerprinting so as to obtaingenomic profiles to compare for diagnostic and/orepidemiological and/or classification purposes.Moreover, for this type of research it is not necessaryto have great quantities of DNA or it is indispensable tohave a high grade of purity, so the yield differencebetween the two methods is not important.Thus, we can conclude that the use of magneticbeads greatly facilitates research in the mycologicalfield where PCR-ready DNA must be available, makingit possible even in microbiological laboratories withminimum equipment, which allows a certain stan-dardization and homogeneity of the DNA extractionTable 3 DNA mean yield and puritywith the two extraction methods.MycetesDNA extractionPhenol/chloroform DynabeadsYield(lg ml)1) SDPurity(OD 260/OD 280)Yield(lg ml)1) SDPurity(OD 260/OD 280)Candida albicans 6684 1710 1.8 443 235 1.3Cryptococcus neoformans 2022 728 1.4 223 99 1.2Dermatophytes 945 545 1.4 329 256 1.4OD, optical density.1 2 3 4 5 6 7 8 9 10 MFigure 1 Genomic profiles of Candida albicans, Cryptococcus neofor-mans and Microsporum canis strains, obtained from the DNAextracted with the two methods [phenol/chloroform (P/C) andDynabeads]. Lanes: M, DNA size markers; 1–2, Cr. neoformans var.neoformans serotype A extracted by P/C and Dynabeads; 3–4,Cr. neoformans var. neoformans serotype D as above; 5–6, C. albicans23554 as above; 7–8, C. albicans 23870 as above; 9–10, M. canisas above.E. Faggi et al.6  2005 Blackwell Publishing Ltd • Mycoses, 48, 3–7from both yeast and filamentous DNA which in thepast was carried out with the most various andcomplex methods.References1 Morissey DV, Lombardo M, Eldredge JK, Kearney KR,Groody EP, Collins ML. Nucleic acid hybridization assaysemploying dA-tailed capture probes: I. Multiple capturemethods. Anal Biochem 1989; 181: 345–59.2 Augenstein S. Superparamagnetic beads: applicationsof solid-phase RT-PCR. Am Biotechnol Lab 1994; 12:12–4.3 Jiacobsen CS. Microscale detection of specific bacterialDNA in soil with a magnetic capture-hybridization andPCR amplification assay. Appl Environ Microbiol 1995; 61:3347–52.4 Liang BC. 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J Clin Microbiol 2001; 39:3382–5.16 Bougnoux ME, Robert F, Beria S, Cassinat B, Nicolas MH,Dupouy-Camet J. Typage mole´culaire d’isolats de Candidapar random amplified polymorphic DNA. J Mycol Me´d1994; 4: 3–8.17 Bowman BH. A model PCR/probe system for the identifi-cation of fungal pathogens. In: Persing DH, Smith TF,Tenover FC, White TJ (eds), Diagnostic Molecular Micro-biology – Principles and Applications. Washington, DC:American Society for Microbiology, 1993: 423–30.Magnetic beads for fungal DNA extraction 2005 Blackwell Publishing Ltd • Mycoses, 48, 3–7 7

Use of magnetic beads to extract fungal DNA.

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Use of magnetic beads to extract fungal DNA.

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