| 摘要 |
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. |
| 主权项 |
1. A method for detecting mold species in an indoor environment sample, comprising
obtaining total DNA from said sample; amplifying said DNA by a polymerase chain reaction (PCR) using at least one pair of primers comprising sequences specific to a large subunit genomic region (LrDNA region) of said mold species, thereby generating DNA barcode amplicons comprising species-specific DNA sequences from mold species present in said sample; performing an assay comprising hybridization to said amplicons using at least one oligonucleotide probe specific to each of the following mold species: Acremonium strictum, Alternaria alternata, Aspergillus candidus, Aspergillus flavus, Aspergillus fumigates, Aspergillus ochraceus, Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sclerotiorum, Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor, Aureobasidium pullulans, Candida glabrata, Cladosporium cladosporioides, Epicoccum nigrum, Eurotium herbariorum, Fusarium oxysporum, Mucor plumbeus, Paecilomyces variotii, Penicillium purpurogenum, Rhizopus oryzae, Penicillium brevicompactum, Penicillium miczynskii, Ulocladium chartarum, Trichoderma viride, Stachybotrys chartarum, Penicillium chrysogenum, Penicillium spinulosum, Penicillium decumbens, Wallemia sebi, Scopulariopsis candida, Penicillium citreonigrum, Scopulariopsis brevicaulis, Phoma glomerata, and Geotrichum candidum, wherein the oligonucleotide probes are selected from SEQ ID NOS: 109-121, 123-156, and 161-182; and detecting hybridization of the oligonucleotide probes to the barcode amplicons. |
