Identification of putative sequence specific PCR primers for detection of the toxigenic fungal speciesStachybotrys chartarum

Molecular and Cellular Probes (1998) 12, 387–396 Article No. ll980197

Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum R. A. Haugland∗ and J. L. Heckman National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, Ohio 45268, USA (Received 18 May 1998, Accepted 10 August 1998) The nucleotide sequence of a c 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers for S. chartarum in the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo 5.0 software. The automated computer searches were unsuccessful in finding S. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that included S. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for only S. chartarum and one other species of Stachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computergenerated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only for S. chartarum. The relative merits of different PCR strategies for the detection of S. chartarum employing either one or both of the primers identified in this study are discussed.

KEYWORDS: Stachybotrys, internal transcribed spacer, ribosomal RNA, PCR, phylogenetic analysis, primer analysis.

INTRODUCTION

Stachybotrys chartarum (Ehrenberg) Hughes, is a cellulytic saprophyte of apparent world-wide distribution that has been studied for over 50 years in relation to its role in human and animal illnesses. Ingestion of this organism in feedstocks by animals causes a variety of well-characterized clinical, systemic and pathological manifestations collectively referred to as stachybotryotoxicosis.1 The species produces a series

of highly toxic macrocyclic trichothecenes2 that are regarded to be the chemical agents of these effects.3 Anecdotal evidence of human illnesses caused by respiratory exposure to this organism, particularly in agrarian work environments, has been available for some time.1 In recent years, more widespread concern over airborne exposure has resulted from evidence showing that the trichothecene toxins produced by

∗Author to whom all correspondence should be addressed at: National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, Ohio 45268, USA. Tel: +1 513 569 7135, Fax: +1 513 569 7117. E-mail: [email protected]

0890–8508/98/060387+10 $30.00/0

388

R. A. Haugland and J. L. Heckman Table 1.

Fungal cultures

Species, EPA collection number

Source, strain number Geographic origin

Stachybotrys chartarum, 080 Stachybotrys chartarum, 177 Stachybotrys chartarum, 280 Stachybotrys chartarum, 281 Stachybotrys chartarum, 382 Stachybotrys chartarum, 384 Stachybotrys chartarum, 385 Stachybotrys chartarum, 386 Stachybotrys chartarum, 387 Stachybotrys chartarum, 388 Stachybotrys chartarum, 389 Stachybotrys chartarum, 390 Stachybotrys chartarum, 391 Stachybotrys chartarum, 392 Stachybotrys chartarum, 395 Stachybotrys albipes,d 383 Stachybotrys bisbyi, 252 Stachybotrys cylindrospora, 248 Stachybotrys dichroa, 253 Stachybotrys elegans, 249 Stachybotrys kampalensis, 254 Stachybotrys longispora, 258 Stachybotrys microspora, 250 Stachybotrys nephrospora, 255 Stachybotrys oenanthes, 256 Stachybotrys parvispora, 251 Stachybotrys theobromae, 257 Memnoniella echinata, 394 Memnoniella subsimplex, 247

ATCC,a 9182 ATCC, 16026 UAMH,b 6715 UAMH, 7900 ATCC, 26303 ATCC, 48610 ATCC 34915 ATCC, 46994 UAMH, 3228 UAMH, 6417 UAMH, 6425 UAMH, 7568 UAMH, 7598 UAMH, 7720 Air isolatec ATCC, 18873 ATCC, 22173 ATCC, 18851 ATCC, 18913 ATCC, 66760 ATCC, 22705 ATCC, 32451 ATCC, 18852 ATCC, 18839 ATCC, 22844 ATCC, 18877 ATCC, 18905 UAMH, 6594 ATCC, 22699

Washington, DC, USA United Kingdom Saskatchewan, Canada Alberta, Canada Finland Finland Hungary Egypt California, USA Namibia Ontario, Canada British Columbia, Canada Alberta, Canada Alberta, Canada Prince Edward Island, Canada United Kingdom Not disclosed Ontario, Canada United Kingdom New York, USA New Guinea Japan Nigeria Japan Quernsey, United Kingdom Congo Malaysia Alberta, Canada Japan

a

American Type Culture Collection. University of Alberta Microfungus Collection and Herbarium. c Kindly provided by Dr Richard Summerbell, Ontario Ministry of Health. d Perfect state=Melanopsamma pomiformis. b

this organism are present in its respirable conidia4 and also from reports suggesting a relationship between human illnesses and the occurrence of this organism in both domestic and workplace environments.5–7 The ability to recognize S. chartarum may be inportant in many epidemiological studies of building related illnesses that are suspected to be fungi related. Current culture-based methods for detecting indoor air fungi in general, however, may not be effective in detecting this organism. First, it has been observed that while conidia of this organism can grow on many commonly used mycological media, they do not proliferate as rapidly as many other frequently encountered fungal species and hence may be overgrown by these competitors.1 Second, there is evidence that the conidia of S. chartarum rapidly lose viability upon becoming airborne8 without any apparent loss of toxigenicity. These factors can result in either low quantitative estimates or total failure to detect this species in air and clinical samples.9 Studies in the authors’ laboratory have focused on the development of a culture-free, polymerase chain

reaction (PCR)-based method for the detection of S. chartarum. In the current report, two primers directed at the ITS regions of the ribosomal RNA operon are described that are expected to allow the specific detection of S. chartarum DNA in PCR assays. Results from this investigation also shed new light on the possible taxonomic and phylogenetic relationships of species within the currently defined anamorphic genera, Stachybotrys and Memnoniella.

MATERIALS AND METHODS

Cultures and genomic DNA extraction The strains used in this study are listed in Table 1. Species designations of the acquired cultures were verified when possible by microscopic and macroscopic examination of their phenotypic traits10 following growth on cornmeal agar medium (Difco). Mycelial samples (c 10–30 mg) from 2 to 7-day-old

PCR primers for Stachybotrys chartarum

389

(a) NS70 NS71

IT51

IT70 ITS1

18S NS81

5.8S

StacR3

IT80

ITS2

28S StacR4 IT41

IT60

100 bp (b) PCR/Sequencing primers: NS70 NS71 IT51 NS81 IT70 IT80 IT60 NL21

5' -GAGGCAATAACAGGTCTGTGATGC-3' 5' -ACAGGTCTGTGATGCCCTTAGA-3' 5' -GAGGAAGTAAAAGTCGTAACAAGGT-3' 5' -TAATGATCCTTCCGCAGGTTCACC-3' 5' -GGTTCCGGCATCGATGAAGAACG-3' 5' -GCGTTCAAAGATTCGATGATTCAC-3' 5' -CCGCTTCACTCGCCGTTACT-3' 5' -GACTCCTTGGTCCGTGTTTCA-3'

PCR primers: IT41 StacR3 StacR4

5' -GATATGCTTAAGTTCAGCGGGTA-3' 5' -TGCCACTCAGAGAATACTGAAA-3' 5' -CGAGGTCAACGTTCAGAAAGTC-3'

Fig. 1. Map of the rDNA regions sequenced in this study and descriptions of polymerase chain reaction (PCR) and sequencing primers. (a) Target sites of the primers on the rDNA map. Forward primers are shown above the map and reverse primers are shown below. Reverse primer NL21 targets a site in the 28S rRNA gene approximately 550 bp to the right of the region shown. (b) Sequences of the primers. All primers were made on a Model 381A automated DNA synthesizer (Applied Biosystems), purified using disposable reverse phase columns (PE-Applied Biosystems) and quantified on the basis of u.v. absorbance at 260 and 280 nm.

colonies grown on modified Sabauroud dextrose agar (Difco) or other suitable media were harvested with a spatula from the surface of nylon filters (Micron Separations Incorporated) placed over the growth media prior to inoculation. The mycelial samples were dried overnight in a vacuum centrifuge and genomic DNA was recovered by a simplified extraction method involving cell wall disruption by grinding in liquid nitrogen, cell lysis by treatment with a buffer containing SDS and DNA purification by phenol/chloroform extraction and isopropanol precipitation as previously described.11 Portions of the DNA extracts were subjected to agarose gel electrophoresis and ethidium bromide staining. Yields of high molecular weight total DNA (appearing as bands on the 1·5% gels) were estimated by comparisons of their fluorescence signals with those of a series of known mass standards (Gibco/BRL) using a model SI fluorimager (Molecular Dynamics).

PCR amplification and purification of DNA templates for sequencing Polymerase chain reactions contained 200 l NTP’s, 1·5 m MgCl2, 7% glycerol, 500 n NS70 and NL21 or NS71 and NL21 primers (Fig. 1), 0·5–1 ng genomic DNA, 0·9 units ExpandTM High Fidelity PCR System (a mixture of Taq and Pwo DNA polymerases, Boerhinger Mannheim) and the PCR buffer provided with the polymerases. Amplification was performed in a model 480 thermocyler (Perkin Elmer) as follows: DNA, primers and NTP’s (overlayed with mineral oil) were heated at 94° for 5 min and held at 72° during the addition of premixed enzyme, MgCl2, buffer and glycerol; the complete mix was step cycled 30 times at 94° for 1 min, 52° for 15 s and 68° for 4 min with a 7 min, 68° extension following the final cycle. Polymerase chain reaction product yields were quantified from a portion of each reaction by agarose gel

390

R. A. Haugland and J. L. Heckman S. kampalensis S. chartarum M. echinata

99

M. subsimplex S. dichroa

93 95

S. oenanthes S. cylindrospora S. microspora

74

S. nephrospora S. albipes

94

S. theobromae

73 100

95

S. bisbyi S. elegans

97

S. longispora S. parvispora Fusarium sambucinum

Fig. 2. Phylogenetic relationships of Stachybotrys, Memnoniella and Fusarium species inferred by the Neighbor-Joining method from the nucleotide sequences determined in this study. The scale bar indicates a distance of 0·01 (one base change per 100 nucleotide positions). Values appearing above the branches are percentages of 1000 bootstrap analysis replicates in which the branches were found. Only percentages greater than 50 are shown. The S. chartarum sequence used in this analysis was that of ATCC strain 9182 (EPA collection #080). The Fusarium sambucinum sequence was designated as an outgroup in the analysis; however, this is an unrooted tree.

electrophoresis as detailed above. The remainder of the samples were subjected to three cycles of dilution with distilled water and concentration by centrifugation in Centricon 100 concentrators (Millipore Corporation) according to vendor instructions to remove unincorporated primers.

precipitation following protocols provided by the vendor of the sequencing kits. Electrophoresis and automated analyses of sequence ladders were performed using a model 373A DNA sequencer (PE/ Applied Biosystems). Compilation and editing of the multiple sequences generated from each template were performed using the SeqManTM software program (PE/Applied Biosystems).

DNA sequencing Sequencing of double-stranded PCR product templates was performed using either Taq DyeDeoxyTM terminator cycle sequencing kits or ABI PRISMTM dye terminator cycle sequencing kits (PE/Applied Biosystems) according to vendor instructions. The sequencing strategy and primers used are shown in Fig. 1a and Fig. 1b, respectively. Purification of the extension products was performed by either a phenol/ chloroform extraction procedure or by direct ethanol

Sequence alignment and phylogenetic analysis Compiled sequences were aligned with the MultAlin 4.0 software program (F. Corpet, Centre de Recherches, I.N.R.A. de Toulouse, France) using default parameters. A minimal amount of manual editing was performed based on visual inspection of the alignment. Evolutionary distances were calculated with the elimination of positions containing gaps

PCR primers for Stachybotrys chartarum

391

ITS region sequences of S. chartarum and all non-target species

Step 1 Automated computer searches for primers using Oligo 5.0

No primers found

Step 2 Phylogenetic analysis

Sequences of species most closely related to S. chartarum removed from search list

Step 3 Automated computer searches for primers using Oligo 5.0

StacR4

Step 4 Manual inspection of aligned sequences

S. chartarum—specific sequences identified

Step 5 Primer performance and primer efficiency analyses using Oligo 5.0

StacR3

Fig. 3. Flow diagram of search and analysis procedures used in this study for the identification of sequence specific polymerase chain reaction (PCR) primers for Stachybotrys chartarum and closely related species.

using the Jukes-Cantor method option in the DNADIST program of PHYLIP v. 3.5.12 A phylogenetic tree was constructed from these distances using the Neighbor-Joining method option in the NEIGHBOR program of the same software package. Support for the branches was estimated by bootstrap analysis of 1000 data sets.13 Phylogenetic trees were also constructed based on parsimony analysis using both the heuristic and branch and bound search options in PAUP v. 3.0.14 Nucleotide substitutions were equally weighted and unordered, and alignment gaps were treated as missing information in these analyses.

Identification of sequence-specific PCR primers Primer searches were performed both by computer and by manual inspection of the aligned sequences. Computer searches were conducted using an automated search feature in the Oligo v. 5.0 primer analysis software package (National Biosciences, Inc.) that eliminates oligonucleotides with adverse primer performance characteristics such as hairpin structures, homo-oligomers, absence of a GC clamp, highly stable 3′ termini and the potential for duplex and primer-dimer formation. These searches also incorporated a priming efficiency analysis algorithm

that automatically eliminates oligonucleotides predicted to undergo false priming of user-specified nontarget sequences (e.g. sequences from other organisms). Manually identified primer candidate sequences were also individually evaluated for predicted PCR performance and specificity using the same analysis features incorporated in the automated searches described above.

PCR primer performance analyses Polymerase chain reactions were prepared and carried out as described above with the following differences: reactions contained 106 or 108 copies of purified NS70-NL21 or NS71-NL21 PCR products as templates (Fig. 1b); AmpliTaq polymerase (Perkin Elmer) was used in place of ExpandTM High Fidelity PCR System polymerases; reactions contained IT51 as the forward primer and the reverse primers shown at the bottom of Fig. 1b; reactions were subjected to 25 rounds of thermocycling; annealing and extension temperatures were 58°C and 72°C, respectively. Polymerase chain reaction product yields were quantified following agarose gel electrophoresis as described above and staining with SYBR Green I (FMC Corporation). Product quantities were converted from

392

R. A. Haugland and J. L. Heckman

Template source

Template alignment with StacR4

PE Score

Template alignment with StacR3

PE Score

S. chartarum

M. echinata

Not found

M. subsimplex

S. dichroa

S. oenanthes

S. cylindrospora

S. microspora

Not found

S. nephrospora

S. kampalensis

S. albipes

S. theobromae

Not found

S. bisbyi

Not found

S. elegans

Not found

S. longispora

Not found

S. parvispora

Not found

F. sambucinum

N. crassa

Not found

Fig. 4. Computer analysis-based predictions of primer specificities. Ribosomal DNA sequencies of the species indicated at left were searched for sites with homology to the primers StacR4 and StacR3 using the ‘false priming site’ analysis option in Oligo 5.0. Alignments of the primer sequences (upper case) with the most homologous sequences of the rDNA templates (lower case) found in each search are shown. Homologous base pairs in these alignments are indicated by vertical lines (|) and gaps are indicated by hyphens (-). Entries of ‘not found’ denote instances where insufficient homology was found between the primers and the rDNA sequences for the program to make an alignment. Also shown are the priming efficiency (PE) scores generated by the program for each primer-template alignment. These scores were computed using an algorithm that considers the DG stabilities of each of the homologous base pairs in the alignment and their distances from the 3′ end of the primer. Also considered in this algorithm are the number of gaps and mismatches in the alignment and their distances from the 3′ end of the primer. Guidelines set forth in the program manual specify that efficiency scores of equal to or greater than 200 may be predictive of priming activity and these instances are annotated with a plus sign (+). Primers IT51 and IT41 were 100% homologous with all rDNA sequences examined and produced priming efficiency scores of 472 and 483, respectively.

PCR primers for Stachybotrys chartarum

units of mass to copy numbers based on the molecular weights of the DNA fragments for the calculation of amplification step efficiencies.

Nucleotide sequences and alignment Nucleotide sequences determined in this study have been submitted to the GenBank database (accession numbers AF081468-AF081483). The Fusarium sambucinum sequence used for phylogenetic analysis is a composite sequence derived from the 18S, ITS1, 5.8S, ITS2 and 28S rDNA sequences of NRRL strain 13708 (kindly provided by Dr K. O’Donnell, USDA, ARS, Peoria, IL, USA, GenBank accession numbers AF081467, X65480 and X65474). The GenBank accession number of the Neurospora crassa sequence used for primer specificity analysis is M13906. Copies of the sequence alignment generated in this study can be obtained from the corresponding author upon request.

RESULTS Sequence variation among the different S. chartartum strains examined in this study was limited to a single position located 58 bases into the ITS 2 region. Strains 080, 386, 387, 388, 389 and 392 (EPA collection numbers, see Table 1) contained a ‘T’ at this position whereas the remaining strains contained a ‘C’. These results indicate a high degree of sequence conservation within ITS regions of this species and consequently primers or probes directed at these regions can be expected to detect most, if not all, strains from the environment. In contrast, substantial sequence differences were found between S. chartarum and all other species of Stachybotrys and Memnoniella in both the 18S rRNA gene and the ITS regions. JukesCantor pairwise distances between the S. chartarum sequence and those of the other species ranged from 3·41 to 7·12% (9·56% for the outgroup species, Fusarium sambucinum), based on the authors’ alignment of the sequences. A phylogenetic tree was constructed by the Neighbor-Joining method from pairwise distances recalculated after the exclusion of alignment positions containing gaps (Fig. 2). S. chartarum occurred in a strongly supported clade that also included S. kampalensis, S. dichroa, S. oenanthes, S. cylindrospora, S. microspora, S. nephrospora, Memnoniella echinata and M. subsimplex. This clade was also found in the most parsimonious trees obtained from both heuristic and branch and bound searches using PAUP (data not shown) with similar support (88% of 1000 bootstrap

393

replicates using the heuristic search option). The species clustering indicated by this tree is inconsistent with the current classification of Stachybotrys and Memnoniella as distinct genera which is based primarily on differences in conidia formation.10 This tree is more consistent with a previously proposed classification system that distinguishes hyaline species of Stachybotrys such as S. bisbyi and S. elegans from the other species which are pigmented.15 An overview of the search strategy employed in this study to identify PCR primers for S. chartarum is presented in Fig. 3. Step 1 in this process was comprised of several different computer searches using the Oligo 5.0 program, including one for sequencespecific primer pairs (i.e. where both primers are specific for the S. chartarum target sequence), one for specific forward primers and one for specific reverse primers. Each of these searches failed to identify suitable primers. As illustrated in steps 2 & 3 of Fig. 3, the phylogenetic analyses of Stachybotrys and Memnoniella species performed in this study, led to further computer searches which excluded the species most closely related to S. chartarum (i.e. those occurring in the same phylogenetic clade as indicated above). These searches identified the group-specific reverse primer StacR4 (Fig. 1b). This primer targets a sequence spanning the boundary of the ITS2 region and 28S rRNA gene (Fig. 1a) which was found to be fully conserved in most of the species occurring in the clade with S. chartarum (Fig. 4). Priming efficiency (PE) scores determined by the Oligo 5.0 software predicted that this primer would also prime DNA from the two species in this clade with heterologous sequences (i.e. S. nephrospora and S. kampalensis, Fig. 4). As illustrated in step 4 of Fig. 3, a manual search of the ITS regions in the sequence alignment resulted in the identification of several oligonucleotide sequences which appeared to be unique to S. chartarum. Further analyses of these sequences using the Oligo 5.0 software, as illustrated in step 5 of Fig. 3, resulted in the identification of the reverse primer StacR3 (Fig. 1b) which targets a sequence located in the ITS1 region (Fig. 1a). The PE scores determined by the Oligo 5.0 software (Fig. 4) predicted that this primer would undergo priming of sequences only from S. chartarum and S. cylindrospora. It was also noted, however, that the PE score of this primer with the S. cylindrospora sequence, was only slightly above the threshold for priming specified by the software guidelines (Fig. 4). Experimental analyses of the PCR amplification performances of StacR4 and StacR3, in conjunction with the universally conserved forward primer IT51, were conducted and the results are illustrated and summarized in Fig. 5. Under the conditions employed

394

R. A. Haugland and J. L. Heckman (a) Lane:

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17

2 kb/50 ng 1.2 kb/30 ng 0.8 kb/20 ng 0.4 kb/10 ng 0.2 kb/5 ng 0.1 kb/2.5 ng PE:

447 447 447 447 447 447 447 285 223 33 33

ASE:

0

0

33

0

0.65 0.66 0.67 0.67 0.66 0.66 0.64 0.38 0.16 0.16 0.16 0.16 0.16 0.16 0.16

SD:

0.02 0.02 0.03 0.02 0.01 0.01 0.05 0.02

*

*

*

*

*

*

*

(b) Lane:

1

2

3

4

426 44

0

5

6

7

8

9

10 11 12 13 14 15 16 17

39 89 203 1

0

15 69

2 kb/50 ng 1.2 kb/30 ng 0.8 kb/20 ng 0.4 kb/10 ng 0.2 kb/5 ng 0.1 kb/2.5 ng PE: ASE: SD:

0 158 14

0

71

0.56 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.05

*

*

*

*

*

*

*

*

*

*

*

*

*

*

Fig. 5. Gel electrophoresis and fluorescence imaging of polymerase chain reaction (PCR) products from amplifications containing the primers StacR4 or StacR3. Polymerase chain reactions were performed as described in the Materials and Methods section using IT51 as the forward primer and (a) StacR4 or (b) StacR3 as reverse primers. Lane 1 in both (a) and (b) contains DNA standards with sizes and mass quantities indicated at left. Lanes 2–17 in both (a) and (b) were loaded with 5 ll aliquots of 50 ll PCRs initiated with 106 template copies from the following species: lane 2, Stachybotrys chartarum (EPA collection #080); lane 3, Memnoniella echinata; lane 4, M. subsimplex; lane 5, S. dichroa; lane 6, S. oenanthes; lane 7, S. cylindrospora; lane 8, S. microspora; lane 9, S. nephrospora; lane 10, S. kampalensis; lane 11, S. albipes; lane 12, S. theobromae; lane 13, S. bisbyi; lane 14, S. longispora; lane 15, S. parvispora; lane 16, S. elegans; lane 17, no template control. The gel was stained with SYBR green I and scanned with a fluorimager. Computer generated primer efficiency (PE) scores and experimentally determined amplification step efficiency (ASE) values for each primer/template combination are superimposed below the respective lanes in the image. Amplification step efficiencies were calculated by the equation: [Sn/So)−1]1/n, where n=number of amplification cycles, Sn=copy numbers of amplification products and So= copy numbers of starting templates. Amplification step efficiency values with standard deviations () shown below are averages from a minimum of three replicate experiments. Lanes with an asterisk (∗) in place of a  value denote primer-temple combinations from which no PCR products were detected in amplifications containing either 106 or 108 starting template copies. The ASE values shown in these lanes are the lowest values that would be measurable based on the product detection limits of the gel staining assay.

in these experiments, reactions containing the IT51StacR4 primer set and templates from the different species with target sequences that were fully homologous with StacR4 (i.e. S. chartarum, M. echinata, M. subsimplex, S. dichroa, S. oenanthes, S. cylindrospora and S. microspora) in each case generated strong product bands as determined by gel electrophoresis (Fig. 5a). Product bands of similar intensity were obtained from the templates of all species examined in this study when the universally conserved

reverse primer IT41 was used in combination with IT51 under the same conditions (data not shown). The average amplification step efficiencies of these reactions were estimated to be 0·66 (=0·01) and 0·7 (=0·02) for the IT51-StacR4 and IT51-IT41 primer sets, respectively. Consistent with PE score predictions, the heterologous template from S. nephrospora was also amplified by the IT51-StacR4 primer set, albeit with a significantly reduced estimated step efficiency of 0·38 (=0·2). In apparent

PCR primers for Stachybotrys chartarum

disagreement with PE score prediction, the template from S. kampalensis was not measurably amplified by this primer set. Amplifications containing the IT51-StacR3 primer set and the homologous template from S. chartarum also generated readily discernable product bands (Fig. 5b), although their gel staining intensities were significantly less than those produced by the IT51-StacR4 and IT51-IT41 primer sets under the same conditions. While the smaller size of the IT51-StacR3 product (198 bp compared with c 558 and 594 bp for the IT51-StacR4 and IT51-IT41 products, respectively) would contribute to this observation, estimations of the step efficiencies of these reactions gave an average value of only 0·56 (: 0·05). This lower amplification efficiency was consistent with the lower PE score of StacR3 with its homologous template (Fig. 4). A second apparent instance of an inaccurate PE score prediction was observed in the failure of the IT51StacR3 primer set to measurably amplify the template from S. cylindrospora.

DISCUSSION Results from this study illustrate both the utility and some apparent weaknesses of the Oligo 5.0 software package in searching for and evaluating sequencespecific PCR primers against a background of related non-target sequences. This experience demonstrates that prior determinations of the overall similarities of the sequences under investigation (e.g. phylogenetic analyses) can be useful in designing appropriate strategies for performing automated searches with this software. Nevertheless, the automatic search feature was unsuccessful in identifying a specific primer for S. chartarum against the background of sequences from its nearest relatives. The failure to identify StacR3 in this search stemmed from the stringency threshold imposed by the software for eliminating oligonucleotides with predicted false priming activities. Oligos with PE scores of 200 or greater for any of the non-target templates included in a search are automatically discarded in this process. The experimental results of this study suggest that this imposed threshold may be somewhat lower than necessary since neither the S. cylindrospora/StacR3 nor the S. kampalensis/StacR4 template-primer combinations (with PE scores of 203 and 233, respectively) were found to generate amplification products. While these results do not preclude the possibility that some priming does occur in these instances, the rates would be presumably too low to be of consequence in most analyses. Aside from these two exceptions, all PE score predictions were found to be both qualitatively

395

and quantitatively consistent with experimental results in this study. These results indicate that the use of StacR3 in conjunction with a non-discriminatory forward primer such as IT51 should be sufficient for the specific detection of S. chartarum sequences in a PCR assay. The potential sensitivity of such an assay could be diminished, however, by the somewhat low amplification efficiency of StacR3. An alternative strategy that is suggested by the results would be a nested or semi-nested PCR, incorporating StacR4 as the reverse primer in the first amplification stage and StacR3 in the second. Such an approach can be expected to increase the detection limits of the assay16 and would also provide additional assurance of specificity when dealing with complex biological samples. A third strategy would be to perform one-step amplifications utilizing StacR4 and to probe any resultant PCR products with the StacR3 sequence. This approach would also benefit from both the higher amplication efficiency of StacR4 and the higher discriminatory power of StacR3 but would eliminate the need for a nested PCR. This laboratory is currently investigating a modification of this approach using the TaqManTM fluorogenic sequence detection system. This system should allow for the confirmational detection of S. chartarum PCR products without the need for followup conventional hybridization analyses.17

ACKNOWLEDGEMENTS The research of J.L.H. was supported in part by an appointment to the Postgraduate Research Participation Program administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US DOE and the US EPA.

REFERENCES 1. Forgacs, J. (1972). Stachybotryotoxicosis. In Microbial Toxins, Vol. VIII. (Kadis, S., Ciegler, A. & Ajl, S. J., eds) pp. 95–128. New York: Academic Press. 2. Jarvis, B. B., Eppley, R. M. & Mazzola, E. P. (1983). Chemistry and bioproduction of the macrocyclic trichothecenes. In Trichothecenes: Chemical Biological and Toxicological Aspects, Vol. 4 (Ueno, Y., ed.) pp. 20–38. Amsterdam: Elsevier. 3. Eppley, R. M. & Bailey, W. J. (1973). 12,13,-Epoxy-D9trichothecenes as the probable mycotoxins responsible for stachybotryotoxicosis. Science 181, 758–60. 4. Sorenson, W. G., Frazer, D. G., Jarvis, B. B., Simpson, J. & Robinson, V. A. (1987). Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra. Applied and Environmental Microbiology 53, 1370–75. 5. Croft, W. A., Jarvis, B. B. & Yatatwara, C. S. (1986). Airborne outbreak of trichothecene toxicosis. Atmospheric Environment 20, 549–52.

396

R. A. Haugland and J. L. Heckman

6. Johanning, E., Morey, P. R. & Jarvis, B. B. (1993). Clinical-epidemiological investigation of health effects caused by Stachybotrys atra building contamination. In Indoor Air ’93. Proceedings of the Sixth International Conference on Indoor Air Quality and Climate, Vol. 1. Health Effects. Indoor Air ’93, Helsinki, pp. 259–263. 7. Dearborn, D. G. (1997). Pulmonary hemorrhage in infants and children. Current Opinion in Pediatrics 9, 219–24. 8. Kozak, P. P. Jr., Gallup, J., Cummins, L. H. & Gillman, S. A. (1980). Currently available methods for home mold surveys. II. Examples of problem homes surveyed. Annals of Allergy 45, 167–76. 9. Flannigan, B. & Miller, J. D. (1994). Health implications of fungi in indoor environments—an overview. In Health Implications of Fungi in Indoor Environments. Air Quality Monographs, Vol. 2. (Samson, R. A., Flannigan, B., Flannigan, M. E., Verhoeff, A. P., Adan, O. C. G. & Hoekstra, E. S., eds) pp. 3–28. Amsterdam: Elsevier. 10. Jong, S. C. & Davis, E. E. (1976). Contribution to the knowledge of Stachybotrys and Memnoniella in culture. Mycotaxon 3, 409–85. 11. Weising, K., Nybom, H., Wolff, K. & Meyer, W. (1995).

12.

13.

14.

15.

16.

17.

DNA Fingerprinting in Plants and Fungi. Boca Raton: CRC Press. Felsenstein, J. (1991). PHYLIP (Phylogenetic Inference Package) Version 3.57c. Computer programs distributed by the Department of Genetics. Seattle, WA: University of Washington. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–91. Swofford, D. L. (1991). PAUP: Phylogenetic Analysis Using Parsimony, Version 3.0. Computer program formerly distributed by the Illinois Natural History Survey. Champaign, IL. Srinivasan, K. V. (1958). Fungi of the rhizosphere of sugarcane and allied plants. I. Hyalostachybotrys gen. nov. Journal of the Indian Botanical Society 37, 334– 342. Stark, K. D. C., Nicolet, J. & Frey J. (1998). Detection of Mycoplasma hypopneumoniae by air sampling with a nested PCR. Applied and Environmental Microbiology 64, 543–8. Heid, C. A., Stevens, J., Livak, K. J. & Williams, P. M. (1996). Real time quantitative PCR. Genome Research 6, 986–94.