Microarray-Based Multiplex Fungal Pathogen Analysis

A method for quantitating at least one fungus in an agricultural product, comprising:

• a) obtaining a sample of the agricultural product;
• b) isolating total nucleic acids from the sample;
• c) performing on the total nucleic acids an asymmetric PCR amplification reaction using at least one fluorescent labeled primer pair comprising an unlabeled primer, and a fluorescently labeled primer, selective for a target nucleotide sequence in the fungus to generate at least one fluorescent labeled fungal amplicon;
• d) hybridizing the fluorescent labeled fungal amplicons to a plurality of nucleic acid probes each having a sequence corresponding to a sequence determinant in the fungus, each of said nucleic acid probes attached at a specific position on a solid microarray support;
• e) washing the microarray at least once;
• f) imaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled fungal amplicons;
• and
• g) calculating an intensity of the fluorescent signal, said intensity correlating with a quantity of the fungus in the sample, thereby quantitating the at least one fungus in the agricultural product.