JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/01/$04.0010 DOI: 10.1128/JCM.39.6.2261–2266.2001

June 2001, p. 2261–2266 Vol. 39, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Identification of Aspergillus fumigatus and Related Species by NestedPCR Targeting Ribosomal DNA Internal

Transcribed Spacer RegionsJUN ZHAO, FANRONG KONG, RUOYU LI,* XIAOHONG WANG, ZHE WAN,

AND DUANLI WANG

First Hospital and Research Center for Medical Mycology of Peking University,Peking University, Beijing, People’s Republic of China

Received 28 August 2000/Returned for modification 2 January 2001/Accepted 7 March 2001

Aspergillus fumigatus is the most common species that causes invasive aspergillosis. In order to identifyA. fumigatus, partial ribosomal DNA (rDNA) from two to six strains of five different Aspergillus species wassequenced. By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNAinternal transcribed spacer (ITS) regions of A. fumigatus. A nested PCR method for identification of otherA. fumigatus-related species was established by using the primers. To evaluate the specificities and sensitivitiesof those primers, 24 isolates of A. fumigatus and variants, 8 isolates of Aspergillus nidulans, 7 isolates ofAspergillus flavus and variants, 8 isolates of Aspergillus terreus, 9 isolates of Aspergillus niger, 1 isolate each ofAspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizuton-gii, Aspergillus beijingensis, and Exophiala dermatitidis, 4 isolates of Candida, 4 isolates of bacteria, and humanDNA were used. The nested PCR method specifically identified the A. fumigatus isolates and closely relatedspecies and showed a high degree of sensitivity. Additionally, four A. fumigatus strains that were recentlyisolated from our clinic were correctly identified by this method. Our results demonstrate that these primersare useful for the identification of A. fumigatus and closely related species in culture and suggest further studiesfor the identification of Aspergillus fumigatus species in clinical specimens.

Invasive aspergillosis is associated with a high rate of mor-tality among patients receiving bone marrow transplantationsor those with leukemia, other cancers, or respiratory ailments.Medical advances that predispose patients to invasive aspergil-losis include intravenous catheterization and treatment withimmunosuppressive drugs, radiation, and high doses of corti-costeroids, among others (2, 5, 24, 28). Early diagnosis andinitiation of antifungal therapy are therefore essential to re-duce the high rate of mortality. Traditional diagnostic methodsused in the clinical laboratory include microscopy, culture, orantigen detection. However, these methods are time-consum-ing, have low degrees of sensitivity, are difficult to standardize,and/or are nonspecific (3, 6, 7, 13, 14). Furthermore, misiden-tification can usually occur because some fungi may be poorlycharacterized by classical techniques or by the inexperiencedperson. Delays in diagnosis may impede the selection of anti-fungal agents when identification of the pathogen is required(21, 23). PCR-based techniques that target DNA have avoidedmany of these problems and provide an alternative approach.PCR-based assays have recently been adapted to the detectionand identification of pathogenic Aspergillus spp. The high de-grees of sensitivity and specificity of these methods provide amethod for the early diagnosis of invasive aspergillosis (6, 7,10, 12). The goal of our study was to develop a practical, quick,

cheap, and specific nested PCR method for the identificationof A. fumigatus, the most common of the Aspergillus pathogens.

MATERIALS AND METHODS

Organisms and culture. Twenty-four isolates of A. fumigatus and variants,eight isolates of Aspergillus nidulans, seven isolates of Aspergillus flavus andvariants, eight isolates of Aspergillus terreus, nine isolates of Aspergillus niger, oneisolate of each of Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus ver-sicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis, andExophiala dermatitidis, four isolates of Candida, four isolates of bacteria, andhuman DNA were used. All molds were initially identified by morphology and allyeasts were identified by fermentation and assimilation tests with the API 20CAUX system (Table 1). The bacterial type cultures were provided by The QualityControl Center for Clinical Laboratory, Ministry of Health (Beijing, People’sRepublic of China). All filamentous fungi were inoculated onto potato dextroseagar slants (Difco, Detroit, Mich.) at 27°C for 72 to 120 h and were stored at 4°Cuntil needed. Yeasts were inoculated in yeast extract-peptone-glucose broth (1%yeast extract, 2% Bacto Peptone, 1% glucose; Difco) and were shaken at 27°C for18 h. Bacteria were inoculated into Luria-Bertani medium (Bacto-Tryptone, 10g/liter; Bacto Yeast Extract, 5 g/liter; NaCl, 10 g/liter [pH 7.0]) and were shakenfor 18 h at 27°C.

DNA preparation. (i) Fungal DNA isolation methods were adopted as de-scribed previously (29). Briefly, mycelia were gently removed from cultures, whileyeasts were pelleted in a 1.5-ml Eppendorf tube. Five hundred microliters ofextraction buffer (100 mM Tris-HCl [pH 9.0], 40 mM EDTA), 60 ml of 20%sodium dodecyl sulfate, and 300 ml of benzyl chloride were added to each sample.The reaction mixture was vortexed and incubated in a 50°C water bath for 40 minand then shaken for 10 min so that the two phases were mixed thoroughly. Then,60 ml of 3 M sodium acetate (pH 5.0) was added, and the tube was kept on icefor 20 min. After centrifugation at 3,500 3 g at 4°C for 15 min, the supernatantwas collected and DNA was precipitated with isopropanol (1:1). The DNA pelletwas resuspended in 300 ml of TE buffer (10 mM Tris-HCl [pH 7.4]–1 mMEDTA), and 1.5 ml of RNase (10 mg/ml) was added. After 5 min, the sampleswere extracted with phenol-chloroform (1:1 [vol/vol]) and, following chloroformextraction, were precipitated with isopropanol. The DNA pellet was resuspendedin 200 ml of TE buffer, and 3 to 5 ml was electrophoresed. All fungal DNAsamples were purified with the DNA Rapid Purification kit (BioDev, Beijing,

* Corresponding author. Mailing address: First Hospital and Re-search Center for Medical Mycology of Peking University, PekingUniversity, No. 8 Xishiku St., West District, Beijing 100034, People’sRepublic of China. Phone: (86-10)6617 1122-3056. Fax: (86-10)66518714. E-mail: lrywdh@public.bta.net.cn.

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People’s Republic of China) after DNA extraction. (ii) Extraction of DNA frombacteria and peripheral blood from healthy humans was done by previouslydescribed procedures (25), with minor modifications.

DNA sequencing. The fragments containing the internal transcribed spacerregions 1 and 2 (ITS1 and ITS2) of the ribosomal DNA (rDNA) complex were

amplified with previously published (8, 20) panfungal primers, primers ITS1,ITS2, ITS3, and ITS4 (ITS1, 59-TCC GTA GGT GAA CCT GCG G-39; ITS2,59-GCT GCG TTC TTC ATC GAT GC-39; ITS3, 59-GCA TCG ATG AAGAAC GCA GC-39; ITS4, 59-TCC TCC GCT TAT TGA TAT GC-39) (amplifi-cation conditions are described below). The PCR product was purified with a

TABLE 1. Organisms used in the study and results of nested PCR

Organism andBMU no.a Originb q-PCR

resultcs-PCRresultd

n-PCRresulte

Organism andBMU no. Origin q-PCR

results-PCRresult

n-PCRresult

A. fumigatus00308 CBM-FD-139 NT 1 100309 UA97-675 NT 1 100311 UA97-609 NT 2 100312 UA97-602 NT 2 100313 CBM-FD-137 NT 2 100514 C NT 2 100315 Clin NT 2 100572 f C4321 NT 2 100574 f C4459 NT 2 100581 env NT 1 100582 env NT 1 100584 env NT 1 100586 env NT 1 100593 f T2363 NT 1 100594 f T2400 NT 2 100595 f env NT 2 101200g Clin NT 2 101340g Clin NT 2 101887g Clin NT 2 101910g Clin NT 2 1

A. fumigatus var.neoellipticus00624 f C0093 NT 2 1

N. fischeri var. fischeri001124 NF.F1 1 1 1001129 NF.F3 1 2 1

N. fischeri var. spinosa001130 NF.S4 1 1 1001131 NF.S7 1 2 1

N. fischeri var. glabra001132 NF.G2 1 2 1001133 NF.G8 1 2 1

A. brevipes 00320 CBM-FD-085 1 2 1

A. nidulans00354 CBM-FA-094 1 2 200356 f UA97-726 1 2 200357 CBM-FA-660 1 2 200358 env 1 2 200359 IFM42011 1 2 200477 CBM-FA-664 1 2 200643 f Unknown 1 2 200660 Unknown 1 2 2

A. flavus00327 CBM-FD-096 1 2 200328 CBM-FD-100 1 2 200534 env 1 2 200541 env 1 2 200599 f C1931 1 2 2

a BMU, Beijing Medical University (now the Peking University Health Science Center).b CBM, National History Museum & Institute, Chiba, Japan, UA, University of Texas Health Science Center at San Antonio; Clin, clinical isolate; env, environmental

isolate; T, Tongren Hospital, Beijing, People’s Republic of China; C, Institute of Microbiology of Chinese Academy of Science; Cen, Center for Quality Control onClinical Chemistry, Ministry of Health, Beijing, People’s Republic of China; NF, Pasteur Institute; IFM, Research Center for Pathogenic Fungi & Microbial Toxicoses,Chiba University, Chiba, Japan; IFO, Institute of Fermentation, Osaka, Japan.

c q-PCR, quality control; some of the isolates were tested with primers ITS1 to ITS4, and the others were tested with specific primers (data not shown); NT, not tested.d s-PCR, single PCR.e n-PCR, nested PCR.f Strains being sequenced for primer design.g Strains used in clinical isolate identification study.

00600 f C8467 1 2 201267g Clin 1 2 201606g Clin 1 2 201909g Clin 1 2 2

A. flavus var. columnaris00330

env 1 2 2

A. terreus00321 IFM42059 1 2 200322 CBM-FD-170 1 2 200324 China env 1 2 200326 China clin 1 2 200562 Unknown 1 2 200628 CBM-FD-168 1 2 200630 f C630 1 2 200632 f Clin 1 2 2

A. niger00334 CBM-FD-182 1 2 200336 CBM-FD-153 1 2 200555 env 1 2 200612 f C5334 1 2 200613 f Clin 1 2 200823 Clin 1 2 200619 env 1 2 214824 Unknown 1 2 2Ab8 Unknown 1 2 201633g Clin 1 2 2

A. parasiticus 00333 CBM-FD-159 1 2 2

A. penicilloides 00337 IFO8155 1 2 2

A. versicolor 00338 CBM-FD-174 1 2 2

A. wangduanlii 00376 Clin 1 2 2

A. beijingensis 00377 Clin 1 1 2

A. qizutongii 00378 Clin 1 2 2

CandidaC. albicans 18768 Clin 1 2 2C. tropicalis 18818 Clin 1 2 2C. parapsilosis 19110 Clin 1 2 2C. guilliermondii 00677 Clin 1 2 2C. glabrata 19277 Clin 1 2 2

E. dermatitidis 00039 Clin 1 2 2

BacteriaEscherichia coli Cen NT 2 2Pseudomonas aeruginosa Cen NT 2 2Staphylococcus aureus Cen NT 2 2Streptococcus fimicarius Cen NT 2 2

Blood 2 2 2

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commercial purification kit and sequenced in both directions by using an ABI373 sequencing machine with Applied Biosystems Taq DyeDeoxy TerminatorCycle-Sequencing Ready Reaction kits according to the manufacturer’s instruc-tions. The isolates sequenced included two isolates each of A. flavus, A. nidulans,A. niger, and A. terrus and six isolates of A. fumigatus (Table 1). Another isolateof A. fumigatus (isolate 00582) was also sequenced to verify the specificity of thenested PCR with primers Asp1 and AFUM1 (Asp1, 59-CGG CCC TTA AATAGC CCG GTC-39; AFUM1, 59-TTA CGA TAA TCA ACT CAG ACT GCATA-39).

PCR amplification. All PCR were the same for each template and set ofprimers used. Each of the reaction mixtures contained 2.5 ml of 103 PCR buffer(100 mM Tris-HCl [pH 9.0] at 25°C, 15 mM MgCl2, 500 mM KCl, 1.0% TritonX-100), 0.5 U of Taq DNA polymerase (Promega), 1 ml of deoxynucleosidetriphosphates (dATP, dCTP, dGTP, and dTTP [10 mM each]; Boehringer Mann-heim GmbH, Mannheim, Germany), 20 pmol of each primer, and 1 ml of sampleDNA. Ultrapure sterile water was added to a final volume of 25 ml. Primers ITS1,ITS2, ITS3, and ITS4 were used for our sequencing amplifications. The DNAsamples used for the nested PCR are indicated in Table 1. When doing thenested PCR, the specific primers used were Asp5 and AFUM2 (Asp5, 59-GATAAC GAA CGA GAC CTC GG-39; AFUM2, 59-ACC TTA GAA AAA TAAAGT TGG GTG-39) in the first amplification. For the second step of the nestedPCR, the products of the first step (1 ml) were used as a template, and primersAsp1 and AFUM1 were used as the specific primers. PCR was performed in aGeneAmp PCR system 9600 instrument (Perkin-Elmer Applied Biosystems,Foster City, Calif.) at 95°C for 5 min for denaturation, 95°C for 30 s for dena-turation, 58°C for 30 s for annealing, and 72°C for 1 min for primer extension for30 cycles, with 5 min of extension at 72°C used for the final cycle. A more rapidscheme may also be used for the identification protocol, in which the reactionconditions are the same as those described above, except that the denaturationand the annealing-extension temperatures are 96 and 70°C, respectively, and thatthe times for the first and second step PCR are 5 and 10 s, respectively.

Multiple sequence alignments and primer design. Multiple sequence align-ments were performed with the Pileup and Pretty programs from the MultipleSequence Analysis program group (provided in Web ANGIS, the 3rd version ofANGIS [Australian National Genomic Information Service]). Specific primerswere designed according to the sequence alignment and reference data (see Fig.1).

Agarose gel electrophoresis. PCR products were analyzed by electrophoresison 2% (wt/vol) agarose gels stained with 0.5 mg of ethidium bromide per ml.Volumes of 10 ml of PCR product and 2 ml of Blue/Orange 63 loading dye wereloaded in each lane. Electrophoretic conditions were 100 V for 45 min in 0.53TAE buffer (Tris-acetate, EDTA electrophoresis buffer [503 concentrated stocksolution which includes, per liter, 242 g of Tris base, 57.1 ml of glacial acetic acid,and 100 ml of 0.5 M EDTA; pH 8.0]). Markers are also run in parallel toapproximate the sizes of the PCR products.

Specificities of the primers. The specificities of the primers were tested withthe organisms indicated in Table 1. Sample DNA was amplified with the appro-priate primers to ensure that they were free of PCR inhibitors. A fragment ofabout 996 bp can be amplified from the positive sample by a single PCR with theouter specific primers, and a fragment of 643 bp can be amplified by the nestedPCR with the inner specific primers. A positive amplicon from A. fumigatus00582 of the expected size (about 643 bp) from a gel was excised, purified, andsequenced as described above for verification.

Sensitivity of the nested PCR. The concentration of DNA from two A. fu-migatus isolates (isolates 00308 and 00586) was determined with a UV spectro-photometer (UV 2100; UV-VIS Recording Spectrophotometer; Shimadzu Co.,Kyoto, Japan) by using 10-fold serial dilutions in TE buffer. The sensitivity of thenested PCR was estimated at the maximal dilution titer by using the A. fumigatus-specific primers.

Isolate identification study. Eight isolates of various Aspergillus species previ-ously identified by morphology methods were selected, inoculated onto Sab-ouraud agar (Oxoid, Hampshire, United Kingdom), and incubated at 35°C for24 h. Four A. fumigatus isolates, three A. flavus isolates, and one A. niger isolatewere used. The samples were coded and presented for processing. A 3-mm2

section (approximately) of mycelium was used for DNA extraction and amplifi-cation. The PCR products were analyzed by agarose gel electrophoresis.

Precautions against contamination and controls. The universal precautionssuggested by Kwok and Higuchi (15) were used to eliminate possible contami-nation of samples. Cross-contamination by aerosols of spores was reduced byusing BSC (Bio-Clean Bench; Sanyo, Tokyo, Japan) or by physical separation oflaboratory areas when we were culturing organisms, isolating sample DNA,preparing PCR mixtures, and analyzing PCR products and by using disposablepipettes. Other precautions included autoclaving of all materials following their

use. The same amplification mixture without template was used as a negativecontrol, and at the same time, positive controls were provided. The fungal DNAswere detected with appropriate primers to verify the identities of the isolates inthe samples and to exclude the existence of PCR inhibitors.

Nucleotide sequence accession numbers. The sequence (ITSI-5.8S-ITS2 re-gion) acession numbers for the isolates (in parentheses) are as follows: AF07890(00593), AF07891 (00594), AF07892 (00595), AF109329 (00572), AF19330(00574), AF109328 (00624), AF07894 (00599), AF07893 (00600), AF07899(00356), AF07898 (00643), AF07897 (00632), AF07896 (00630), AF07895(00613), and AF109327 (00612). The accession numbers for the other sequencesobtained from GenBank for use with primers designed from small-subunit rRNAand ITS1-5.8S-ITS2 (in parentheses) are as follows: A. fumigatus, AB008401,M55626, and M60301 (AF07889); A. flavus, X78537 and D63696 (AB008414);A. nidulans X78539, AB008403, and U77377 (U93686); A. terreus, X78540 andAB008409 (AJ001333); A. niger, X78538 and D63697 (U93685); A. parasiticus,D63699 (AB008418 and AF027862); Candida albicans, AJ005123, M60302 andX53497 (L76774, X71088, and L47111).

RESULTS

Multiple sequence alignments and primers. Multiple se-quence alignments were developed from the sequence dataand information from GenBank (Fig. 1). From the multiplesequence alignment results, we noticed that (i) the small-sub-unit, 5.8S, and large-subunit regions from all fungi analyzedare conserved, but small differences are observed; (ii) bothITS1 and ITS2 regions are quite diverse; and (iii) greaterdistinctions between species than between isolates within aspecies are observed (alignment data not show). The ITS1region displayed more interspecies variation than the ITS2region; approximately four separate variable regions wereseen. These results are in agreement with those from otherdetailed studies (11a, 19). We also determined that if universalor genus-specific primers are used, the conserved regions (18S,5.8S, and 28S) are the best targets, but if the specific primersare used, the ITS region is the choice as the target. Therefore,our specific primers were designed from the sequence analysisof the ITS region. The locations of the primers used in thepresent study are indicated in Fig. 1. Upon sequence analysisof ITS1, primer AFUM1 was generated; its sequence is iden-tical to those of all seven A. fumigatus reference isolates testedbut different from those of the other isolates tested. PrimerAFUM2 was generated from the sequence of ITS2; likewise,its sequence was also identical to those in the A. fumigatusisolates tested but different from those of the other isolatestested (data not show). Primer Asp5 (forward) combined withprimer AFUM2 (reverse) as outer primers specific for A. fu-migatus amplified a 996-bp amplicon (approximately);primer Asp1 (forward) combined with primer AFUM1 (re-verse) as inner primers specifically amplified an A. fumigatustarget gene which resulted in a 643-bp amplicon (Fig. 2 and 3).

Precautions against contamination and control. All sampleswere tested with the appropriate primers to rule out false-negative reactions. Actually, in this study we found severalsamples that gave false-negative reactions. However, purifica-tion of the template DNA by using the DNA Rapid Purifica-tion kit overcame this problem except for one isolate of A. niger(isolate 00335). That isolate was eliminated from our study,because amplification was not observed after purification. Nocontamination occurred when we used a negative control.

Specificity of the nested PCR with the designed primers.Seventy-three samples were used in order to evaluate the spec-ificity of the nested PCR with the specific primers designed in

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the present study. The presence of amplicons of about 643 bpon agarose gels as detected under UV light was considered apositive result by nested PCR (Fig. 2). The results are summa-rized in Table 1. The results indicate that the specific primersdesigned for detection of A. fumigatus amplified 16 isolates ofA. fumigatus, 6 isolates of Neosartorya fischeri variants, 1 isolateof A. fumigatus var. neoellipticus, and 1 isolate of Aspergillusbrevipes. Therefore, by using the A. fumigatus-specific primersdesigned in the present study, all isolates of A. fumigatus andclosely related isolates were successfully amplified. These re-sults are in agreement with the sequence data in GenBank, asdetermined with the Basic Blast program with the specificprimers. The amplicon from A. fumigatus 00582 has been se-quenced and compared with A. fumigatus sequences depositedin GenBank. The results show that the amplification positionand length are correct. Using the sequenced amplicon, wefound that our sequence also aligned with the sequences ofA. fumigatus isolates deposited in GenBank. At the same time,no false-positive reactions were found. Interestingly, A. beijin-gensis (17) can be amplified with the outer primers but not withthe inner primers. These results indicate that this new isolate isdistinct at the molecular level from A. fumigatus. Cross-reac-tions with other species of fungi have not been observed.

Sensitivity of the nested PCR. A total of 10 ml of each of thePCR products was loaded onto agarose gels for electrophoresis.The visible bands in the nested PCR were seen at DNA dilu-tions of up to 1:13 to 1:14. This corresponds to 10 to 100 ag ofsample DNA. The nested PCR is therefore more sensitive thanother methods, such as antigen detection or Southern blotting.

Identification of clinical isolates. To determine the utility ofthe nested PCR with primers designed for the accurate iden-tification of A. fumigatus, a blind test was carried out. Eightclinical isolates morphologically confirmed to be A. fumigatuswere tested. Following incubation of the culture plates for 24 hat 35°C and amplification of the isolates by using specific prim-ers, the products were analyzed by agarose gel electrophoresis.By this method, all A. fumigatus cultures were identified cor-rectly to the species level (Fig. 3), and all identifications weremade in less than 12 h after receipt of the culture.

DISCUSSION

Fungi from patient samples need to be correctly identified tothe species level. This information often influences the type,dosage, and duration of antifungal therapy and also providesepidemiological surveillance data (16). A variety of different

FIG. 1. Multiple sequence alignment results for the consensus sequences of the five regions of the rDNA complex. Relevant primers are shown.SSU, small-subunit rRNA gene, partially displayed; ITS1, internal transcribed spacer region 1, completely displayed; 5.8SU, the 5.8S subunit rRNAgene, nucleotides 79 to 148 are omitted; ITS2, internal transcribed spacer region 2, completely displayed; LSU, large-subunit rRNA gene, partiallydisplayed; the dashes in the sequences for the individual species names indicate identity to the consensus nucleotide; dashes in the consensussequence indicate no consensus nucleotide; dots indicate deletion of the nucleotide at that point; shading indicates the positions of the primers.

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PCR strategies have been tried in recent years. Most of thepublished data used 18S rDNA or 28S rDNA as target DNAbut rarely used the ITS regions. However, the resolving power(specificity) is low because of the conservation of these se-quences, so that Aspergillus can be detected only at the genuslevel. Further separation requires an additional, labor-inten-sive step that is also time-consuming (6, 7, 11–13, 20).

Turenne et al. (27) adopted differences in the length of theITS region for the identification of medically important fungi.However, the amplicon lengths were not specific enough to dis-tinguish species, especially when larger samples were used. Al-though sequencing is quite accurate, its labor-intensive natureand expense restrict its use in the clinical laboratory (11a, 27).

The data published thus far indicate that the ITS region issufficiently heterogeneous to differentiate species. Therefore,the ITS region seems to be an excellent target for the deter-mination of species of fungi (11a, 24). Because the primersdetermine the specificity and sensitivity of PCR, they wereextensively considered in the present study. The position andrelationship of the primers to the fungal isolates are shown inFig. 1. By use of the specific primers that target the ITS regionand nested PCR, an easy, cost-effective assay that has a highsensitivity is achieved. The whole procedure can be finishedwithin 4 h if the rapid scheme is used for amplification.

Besides the target gene and primers, the DNA extractionmethod should also be considered carefully. Several papershave detailed progress in improving methods of DNA extrac-tion and also the problems of PCR amplification of fungi (22).Bougnoux et al. (1) reported on an inhibitor of PCR that couldnot be inactivated. In our study, several false-negative reac-tions occurred, but most of them could be resolved after pu-rification of sample DNA with glass milk. One sample of A. ni-ger was eliminated from the analysis because of a negativeresult in the control test. The presence of a PCR inhibitor inthe first step of the PCR in the present study may also havebeen one of the causes of the negative results. Therefore, agood method of extraction of DNA for PCR amplification is animportant step in the identification scheme. Progress in thisfield may lead to breakthroughs in the diagnosis of infections

caused by medically important fungi or the identification of themedically important fungi.

There are many criticisms of PCR for the detection of fun-gal DNA, especially when panfungal primers or nested PCRis used, since molds are so prevalent in the environment. Ina collaborative study among five European centers, the fre-quency and risk of contamination due to airborne sporeinoculation or carryover contamination in fungal PCRs wereanalyzed. The investigators concluded that the risk of con-tamination is no higher in any fungal PCR assays than in oth-er diagnostic PCR-based assays if general precautions aretaken (18). In the present study, the results obtained withthe negative controls demonstrate that contamination didnot occur. Therefore, the incidence of contamination can beavoided if stringent methods are adopted. For the applica-tion of PCR-based strategies to the diagnostic laboratory,we strongly recommend the procedures developed by Bret-agne et al. (4).

Although we have identified A. fumigatus and related spe-cies, there are still some questions that need to be answered.For example, first, is it necessary to identify A. fumigatus to thespecies level? In this study we could not discriminate A. fu-migatus from N. fischeri, a related telemorph species of A. fu-migatus section fumigati and A. brevipes. They are taxonomical-ly included in A. fumigatus section fumigati (9, 16, 28). Ourresults support the belief that they are probably derived froma common ancestor of A. fumigatus. At the same time, they alsodemonstrate the problems associated with the traditional mor-phology-based nomenclature system or whether the target re-gion that we chose for identification is optimal (9, 26) for thesubgrouping of A. fumigatus section fumigati. Second, mostclinical observations identify infectious agents at the genuslevel and samples are studied on a smaller scale with primersor probes. If a larger number of isolates was used, the numbersof samples with false-negative or false-positive results mayincrease due to the great diversity of fungi. It has been re-ported that more than 80% of fungal isolates can be isolatedonly once when tested at the molecular level (5, 28). Somepapers have reported that some cross-reactions exist when

FIG. 2. Results of PCR for specificity of amplification. Lane 1,100-bp ladder; lanes 2, 4, 6, and 8, A. fumigatus 00308, 00309, 00581,and 00586, respectively, lane 3, A. flavus 00327; lane 5, A. niger 00336;lane 7, A. terreus 00321; lane 9, A. nidulans 00359; lane 10, singleamplification of A. fumigatus 00308; lane 11, negative control; lane 12,positive control (isolate 00321). The arrowhead to the left of lane 1indicates a 500-bp marker.

FIG. 3. Results of nested PCR for identification of isolates in clin-ical specimens. Lane 1, 100-bp ladder; lanes 2, 4, 6, 8, A. fumigatus01200, 01340, 01887, and 01910, respectively; lane 3, A. flavus 01276;lane 5, A. flavus 01606; lane 7, A. flavus 01909; lane 9, A. niger 01633;lane 10, known positive isolate (isolate 001124); lane 11, known neg-ative isolate (isolate 00337); lane 12, negative control. The arrowheadto the left of lane 1 indicates a 500-bp marker.

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large number of samples are tested (6). Therefore, the greatdiversity of fungi may be the most significant drawback for thedevelopment of a PCR-based diagnostic method. It seems thatthe problem is not resolvable at present. Third, as we noticedfrom our data and data presented elsewhere, the differences inboth rDNA and mitochondrion genes are sufficient for discrim-ination of fungi at the genus level but are not enough to dis-criminate strains within a species. This problem reflects theneed for more sequence information or even whole-genomesequence analysis. In subsequent years, more work will beneeded to correlate genotypes with the morphologies or phys-iologies of fungi. These results will provide important infor-mation for use in clinical diagnosis.

In conclusion, specific primers with a nested PCR success-fully identified A. fumigatus section fumigati. The assay wasquick, sensitive, and economical. Although there are still prob-lems that need to be addressed before it can be used in clinicallaboratories, we consider this method to be very useful for theidentification of fungi.

ACKNOWLEDGMENTS

Grants from the Ministry of Education of the People’s Republic ofChina supported this study.

We thank Y. Horie, K. Nishimura, Z. T. Qi, Y. X. Wang, J.-P. Latge,and M. Rinaldi for generously providing some of the isolates used inthe study. We also thank D. M. Li for valuable comments on thetaxonomy of Aspergillus. We appreciate R. Calderone’s kind help inreviewing the manuscript.

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