Abstract:
A method based on species-specific molecular markers applied to a multiplex platform system is provided that permits, in a one-time assay, the qualitative and quantitative screening of multi-species populations in several dozens of environmental samples, tested in parallel, with low cost and rapid turn-around. The method identifies a specific portion of DNA, shared amongst a variety of important mold species, but with sufficient sequence differences that allow for unique identifications. Two pairs of primers, which are capable of amplifying this region across at least 38 different mold species, have been identified. Also short oligonucleotide probes have been designed specifically for each species to detect their presence and concentration in mixed species environmental samples. The method of detection of the invention can be practiced using conventional PCR reactions, followed by nucleotide hybridization assays and quantization of the hybridized biotinylated amplicons in a multiplex liquid array system.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims the benefit of U.S. Patent Provisional Application Ser. No. 61/416,800 filed Nov. 24, 2010, the entire content of which is hereby incorporated by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to a method of identifying and quantifying species of indoor fungi in multi-species populations using a specific portion of DNA. The portion of DNA is shared amongst a variety of important indoor mold species, but it is also characterized by sufficient sequence differences that allow for unique identifications. 
       BACKGROUND OF THE INVENTION 
       [0003]    It is widely understood that mold present in living or working spaces, with exposure rates as high as 50% (Gunnbjornsdottir et al.,  Thorax  2006, Mudarri and Fisk,  Indoor Air  2007), presents a human health threat. The toxic nature of mold can lead to a number of health problems both chronic and acute. Various epidemiological studies have revealed that mold exposure is often associated with diseases and conditions such as asthma, respiratory infection, bronchitis, allergic rhinitis, and eczema (Mendell et al.,  Environ Health Perspect  2011). 
         [0004]    As buildings become more energy efficient and better sealed, the potential for increased mold growth is actually exacerbated. As such, the incidence of mold exposure is increasing. Children and elderly people, as well as those with pre-existing respiratory problems are particularly vulnerable. 
         [0005]    There are a number of reasons for routine testing of Indoor Air Quality (IAQ), and in particular to qualify and quantify mold species in indoor environments (mold “testing” vs. general mold “problem identification”). These reasons include the following: 
         [0006]    1) Acceptable levels for individual mold species vary since mold species toxicity differs widely, as does spore size, weight, and other features which affect the risk to building occupants. High priority areas can be identified based on type of molds present to maximize remediation strategies for public buildings (i.e. schools, hospitals, offices) and for private residences. For mold species with severe toxic effects, immediate action from mold professionals is crucial for the safety of the building and its occupants. 
         [0007]    2) The presence of specific group of mold species often indicates building water damage conditions even if it is not visible. Molds often hide behind walls and their presence and level of toxicity can only be indicated by the adequate IAQ tests. 
         [0008]    3) IAQ problems can be identified by comparing known indoor mold species to known outside mold species; which can only be achieved by knowing which species are present. Thus, commonly used tests of total spore counts between the indoor molds and outdoor molds, when mold species are unknown, are largely irrelevant. 4) For a large remediation project exact mold tests can improve project control. By analyzing mold spores qualitatively as well as quantitatively before and after a mold remediation project, it can help ensure that the cleanup is complete. 
         [0009]    5) For house and health insurance purposes. 
         [0010]    The approved ability to assess mold species rapidly and precisely can have an impact on mold remediation and overall health. Method of multiplex mold toxicity assessments described here will help to quickly pin point the sources of mold illness. Improved analysis can lead to a better understanding of mold related illness and prevention. 
       Limitations Of Current Technology 
       [0011]    While physicians can diagnose mold-related symptoms, analyzing the source and degree of mold contamination is currently a difficult and expensive task. 
         [0000]    The tests that have been used so far by the majority of environmental laboratories in order to assess mold contamination and the potential risk to the building occupants are often flawed, time consuming, and costly to customers. 
         [0012]    Currently, there are four basic evaluation methods of mold contamination: 
         [0013]    1) IAQ inspectors simply confirm the presence of visible mold on walls (no species identification and quantification) and mold growth is generally classified by size of the mold colonies on walls; 
         [0014]    2) IAQ inspectors collect air samples and send them to environmental laboratories to assess the total number of spores from visible and hidden mold colonies (without mold species identification and quantification); 
         [0015]    3) Collected air samples are sent to laboratories for viable spore counting from colonies grown on Petri dishes which takes up to 2 weeks (with limited and subjective traditional microscopic species identification and quantification); 
         [0016]    The above three methods are not only, very often, subjective and inaccurate, but also time consuming, inconsistent and not comparable between the labs. 
         [0017]    4) Recently some laboratories have started performing limited molecular analysis of molds with use of qualitative PCR (qPCR) from molds grown at least overnight on Petri dish plates where the mold species from each sample are screened in discrete PCR reactions with the use of pairs of primers specific for each mold species and expensive species-specific fluorescent probes. However, this method offers only low throughput solutions with relatively high cost per sample. 
       SUMMARY OF THE INVENTION 
       [0018]    Mycelia and spores of 38 common indoor fungal species were subjected to molecular analyses in order to obtain species—specific molecular markers. The markers were next applied to the Luminex xmap® liquid array system (Luminex Corporation (NASDAQ: LMNX)). This multiplex nucleic acid assay allows for the identification and quantification of all target mold species which are present in a given environmental sample. With the present assay methodology, at least 38 species of indoor fungi are tested for simultaneously. This invention thus provides a method of multiplexing up to 100 different analytes in parallel, with low cost and rapid profiling (more than 96 samples can be profiled in a 24 hour period). 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0019]      FIG. 1  shows visualization of mold PCR amplicons on 1% agarose gel; the amplicons were obtained from mold mycelia with the use of LrDNA pair of primers 
           [0020]      FIG. 2  shows visualization of mold PCR amplicons on 1% agarose gel; the amplicons were obtained from mold spores with the use of pairs of primers for LrDNA (2 nd  row),) and ITS (3 rd  row). 
           [0021]      FIG. 3  shows a Neighbor Joining phylogenetic tree for indoor mold species in the LrDNA region. 
       
    
    
     DETAILED DESCRIPTION 
       [0022]    Identification in biology is the process of assigning a pre-existing individual or class name to an individual organism. Identification of organisms to individual names (or codes) may be based on individualistic natural body features, experimentally created individual markers (e.g., color dot patterns), or natural individualistic molecular markers. Every living organism contains DNA, RNA, and proteins. The hereditary information of all living organisms (except some viruses) is carried by DNA molecules. In general, closely related organisms have a high degree of homology in the molecular structure of these substances, while the molecules of organisms distantly related usually show a pattern of dissimilarity. 
         [0023]    Molecular identification has been made possible by the availability of techniques for DNA sequencing, which allow the determination of the exact sequence of nucleotides or bases in either DNA or RNA structure which is common to more than one organism. At any location within such a sequence, the nucleotide bases found in a given position may be identical for all members of the same species and may vary between species. The species-specific DNA loci can be identified with the help of the short molecular markers (oligonucleotide or “oligo” probes) which can ferret out a particular sequence of DNA from a group of unknown nucleotides. 
         [0024]    For this invention the large-subunit (LrDNA) in the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA), common to different mold species, was used to obtain PCR amplicons of multiple mold targets and to define species-specific oligo probes. 
         [0025]    The method of detection of the present invention is practiced using one conventional PCR reaction for all mold species in a sample, followed by capture probe microsphere-based hybridization assays containing oligo probes of interest and by quantization of the hybridized biotinylated amplicons in a multiplex liquid array system from Luminex xMAP® technology (Dunbar, Clinica Chimica Acta, 2005 “Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection”). This technology allows dozens of samples to be tested in parallel with highly accurate qualification and quantification of target sequences, low cost per sample, and rapid turn-around. 
         [0026]    The method of the present invention was developed with the use of pure culture slants of the following 38 mold species obtained from the University of Alberta Fungal Collection and from the Canadian Collection of Fungal Cultures/Agriculture and Agri-Food Canada: 
         [0027]    UAMH 10908  Acremonium strictum    
         [0028]    UAMH 10047  Alternaria alternata    
         [0029]    UAMH 7647  Aspergillus candidus    
         [0030]    UAMH 9308  Aspergillus flavus    
         [0031]    UAMH 9311  Aspergillus ochraceus    
         [0032]    UAMH 9312  Aspergillus penicilloides    
         [0033]    UAMH 4247  Aspergillus restrictus    
         [0034]    UAMH 9951  Aspergillus sclerotiorum    
         [0035]    UAMH 7895  Aspergillus sydowii    
         [0036]    UAMH 3627  Aspergillus terreus    
         [0037]    UAMH 9479  Aspergillus ustus    
         [0038]    UAMH 7651  Aspergillus versicolor    
         [0039]    UAMH 10765  Aureobasidium pullulans    
         [0040]    UAMH 10403  Candida glabrata    
         [0041]    UAMH 4146  Cladosporium cladosporioides    
         [0042]    UAMH 10787  Epicoccum nigrum    
         [0043]    UAMH 7767  Eurotium herbariorum    
         [0044]    UAMH 3313  Fusarium oxysporum    
         [0045]    UAMH 8720  Mucor plumbeus    
         [0046]    UAMH 7255  Paecilomyces variotii    
         [0047]    UAMH 3178  Penicillium purpurogenum    
         [0048]    UAMH 4339  Rhizopus oryzae    
         [0049]    UAMH 5145  Penicillium brevicompactum    
         [0050]    UAMH 5150  Penicillium miczynskii    
         [0051]    UAMH 5703  Ulocladium chartarum    
         [0052]    UAMH 6280  Trichoderma viride    
         [0053]    UAMH 6715  Stachybotrys chartarum    
         [0054]    UAMH 6742  Penicillium chrysogenum    
         [0055]    UAMH 6746  Penicillium spinulosum    
         [0056]    UAMH 7817  Penicillium corylophilum    
         [0057]    UAMH 7818  Penicillium decumbens    
         [0058]    UAMH 7897  Wallemia sebi    
         [0059]    UAMH 8405  Scopulariopsis candida    
         [0060]    UAMH 9208  Penicillium citreonigrum    
         [0061]    UAMH 9320  Scopulariopsis brevicaulis    
         [0062]    UAMH 10658  Phoma glomerata    
         [0063]    CCFC225569  Geotrichum candidum    
         [0064]    CCFC226913  Stachybotrys chartarum    
         [0065]    CCFC240363  Aspergillus fumigatus    
         [0066]    The original slants of the pure mold cultures were then used to produce mycelia (cell cultures) for all mold species in the YPD (Yeast Peptone Dextrose) broth cultures and conidiospores for all mold species in the PDA (Potato Dextrose Agar) and/or CER (Cereal Agar) mold cultures. Stocks of conidia were prepared in 5% Tween80 solutions which contained from 2.5×10 4 /mL to 5.4×10 8 /mL of conidiospores. 
       DNA Extraction 
       [0067]    After numerous trials, a method was developed for isolation of the total DNA directly from the conidia of the molds without necessity to grow them into cell cultures. 
         [0068]    The procedure of DNA extraction from mold mycelial cells and their conidia employs a combination of Pro-K enzyme, Elu Quik (Whatman, 10462620)/Sigma (G1N-350) lysing an binding buffers, and the glass bead grinding procedure which is described as follows: 1) glass beads, spore sample, Elu Quik lysing buffer and Elu Quik binding buffer were shaken in a 1.5 mL conical tube for 4 min. and spun at 8,000 xg for 1 min. 
         [0069]    2) supernatant was removed and the remaining material treated with Sigma lysing buffer and Pro-K enzyme and then the whole DNA extracted in the binding columns according to standard Sigma procedure with the use of the GeneElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich Co., St. Louis, Mo., U.S.A.). 
       PCR Conditions 
       [0070]    DNA amplifications from mycelial cells and conidia for all mold species were carried out using two sets of universal primers for isolation of the ribosomal DNA (rDNA) genome: one pair of primers for the large-subunit (LrDNA) region and the second one for the ITS region. 
         [0071]    In the PCR cycling of the LrDNA region universal forward primer F63 (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) (SEQ ID NO:1) and universal reverse primer LR3 (5′-GGT CCG TGT TTC AAG ACG G -3′) (SEQ ID NO: 2) were used to produce DNA amplicons for each of the indoor mold species listed above. 
         [0072]    PCR cycling of the ITS region included universal fungal forward primer ITS5 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′) (SEQ ID NO: 3) and universal fungal reverse primer LR6 (5′-CGC CAG TTC TGC TTA CC-3′) (SEQ ID NO: 4). All PCR reverse primers were biotinylated at the 5′ end to help isolate the biotinylated DNA amplicons of interest by binding them to fluorescent streptavidin. Each PCR cocktail contained 2.3 μL of 10×PCR buffer, pH 8.3 (10 mM of Tris-CHl, pH 8.3; and 50 mM of KCl; 0.01% NP-40), 1.3 μL of 50 mM MgSO 4 , 200 μM of each NTP, 1 unit Taq polymerase, 0.3 μM of each primer, 1-5 μL of DNA template, and the remaining volume of ddH 2 O up to 25 μL. 
         [0073]    The PCR thermal regime consisted of one cycle of 1 min at 95 ° C.; 35 cycles of 1 min at 94° C.; 1 min at 55 ° C.; and 1.5 min at 72 ° C., a final cycle of 7 min at 72 ° C. 
         [0074]    All PCR amplicons were visualized on 1% agarose gels. 
       Sequencing 
       [0075]    All PCR products were subjected to dye terminator cycle sequencing reactions and sequenced on Applied Biosystem&#39;s 3730xl DNA Analyzer technology, using terminators with Big Dye v. 3.1, forward and reverse primers. 
         [0076]    In PCR assays of the LrDNA regions, the 673 by unique sequences (molecular barcodes) were produced for each mold species, as well as, 2234 bp barcodes were produced for 32 mold species for the ITS region. 
         [0077]    Sequences were aligned by employing the Sequencher 4.6 program (Gene Codes Corporation). The phylogenetic analysis of the sequences produced shows that each mold species comprises distinctive sets of LrDNA ( FIG. 3 ) and ITS haplotypes; no sequences are shared among species, while the sequenced individuals from the same species group closely together. 
         [0078]    The present invention has determined that the LrDNA and ITS sequences produced for each indoor mold species show strong phylogenetic signal that can be successfully used in the diagnostics of those species. Probe selection was based on visual sequence alignment of all mold species and areas of sequence divergence among the species were analyzed for probe selection. All probes were designed to be uniform in length (20-mer); some probe lengths were modified to avoid potential secondary structures or an unstable AG, resulting in probe sequences of 20 to 24 bp. The quality of the probe was assessed using the CommOligo program. 
         [0079]    Probes&#39; designs were followed up by the each probe validation, then by their coupling to a different set of 5.6-μm polystyrene carboxylated microspheres using a carbodiimide method (Diaz and Fell, 2004) and multiplex liquid array system from Luminex xMAP® technology. The attached to beads capture probes (complementary in sequence to the biotinylated strands of the target amplicons) were synthesized with a 5′-end Amino C12 modification (IDT, Coralville, Iowa). 
         [0080]    Probes were validated by testing their performance in a multiplex format first of 1-6, then of 6-38 in a capture probe hybridization assays. The capture probes, complementary in sequence to the biotinylated strand of the target amplicon, were synthesized with a 5′-end Amino C12 modification (IDT, Coralville, Iowa). Amplification with the use of biotinylated reverse primers allows for detection by Streptavidin Phycoerythrin; SAPE in Luminex system. 
         [0081]    Each microsphere set used for probe coupling contained unique dye mixture of red and infrared fluorochromes that provided the unique identity of spectral addresses. Coupling reaction involved incubation of probes with microspheres resuspended in 0.1 M MES (2[N-morpholino]ethanesulfonic acid), pH 4.5, with a determined amount of probe (0.1 to 0.4 nmol), followed by incubation with EDC/μl and washing with 0.02% Tween 20 and 0.1% sodium dodecyl sulfate. The beads were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8) and kept in the dark at 4° C. 
         [0082]    Capture probe hybridization assay was based upon detection of 5′-biotin-labeled PCR amplicons hybridized to specific capture probes covalently bound to the carboxylate surface of the microspheres. Hybridization reactions were carried out in 96-well plates in the presence of a 3 M TMAC solution (tetramethyl ammonium chloride-50 mM Tris [pH 8.0]-1 mM EDTA [pH 8.0]-0.1% sodium dodecyl sulfate), consisted of biotinylated amplicons diluted in TE buffer (pH 8) and 1.5× TMAC solution containing a bead mixture of approximately 5,000 microspheres of each set of probes. Prior to hybridization, the reaction mixture was incubated for 5 min at 95° C. in a mastercycler (Brinkman). This step was followed by 15 min of incubation at 55° C. After hybridization, the microspheres were pelleted by centrifugation and the supernatant was removed. The plate was again incubated and the hybridized amplicons were labeled with the fluorescent reporter molecule, streptavidin R-phycoerythrin. 
         [0083]    From the obtained reactions 100 microspheres of each set (100 replicate measurements) were analyzed on the Luminex 100. A blank and a set of positive and negative controls were included in the analyses. MFI (median fluorescence intensity) values were calculated with a digital signal processor and the Luminex proprietary software. Assays and the samples were run in duplicate. The signal-to-background ratio corresponds to the MFI signals of positive controls versus the background fluorescence of samples containing all components except the target DNA. 
         [0000]    The limits of the Luminex system were tested in several assays with the range of quantities from 150 to 10 fmol of biotinylated amplicons in the presence of the reverse and complement oligo probes. The sensitivity of the assays was also estimated using serial dilutions of amplicons (1000 to 10 −3  ng). The quantitative/qualitative method provided by the invention has been developed to detect indoor fungal species in the mixed species environmental samples through: 
         [0084]    1) the use of a new, optimized method of total DNA extraction for indoor mold species; 
         [0085]    2) detection of two specific genomic regions (section of LrDNA region and section of ITS region) which are suitable for obtaining unique molecular barcodes (i.e. genetic profiles) for each of the indoor fungal species; 
         [0086]    3) production of those molecular barcodes in the regular PCR assays with the use of one pair of primers for the LrDNA region and one pair of primers for the ITS region for all 38 mold species listed in the method; 
         [0087]    4) based on their molecular barcodes, designing of one to several molecular probes specific for each of 38 mold species; 
         [0088]    5) adaptation of the Luminex xmap® liquid array system to using those probes for rapid, accurate, and standardized discrimination and quantification of mixed fungal species samples, with no need for growing fungal cell cultures at any time. 
         [0089]    While this invention has been described in connection with the specific examples and methods, it will be appreciated by the skilled artisan that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the ken of the skilled artisan. The claims that follow are not intended to limit the scope of invention in any way, but to define certain essential features of the invention described in detail herein.