Received July 16, 2007

Abstract:

This study employed a microfluidic method in which probe creation does not require pin-spotting and fast hybridization is conducted on the same microarray chip for the detection of three greenhouse pathogens (Botrytis cinerea, Didymella bryoniae, and Botrytis squamosa). In this method, 16 oligonucleotide probe line arrays were created on a glass substrate by a microfluidic printing method. Then, low amounts of the DNA samples (1 fmol of oligonucelotides or 1.4 ng of PCR products) were introduced into the microchannels that were orthogonal to these probe lines. The hybridizations of 16 samples (21-mer complementary oligonuleotides and ~260 bp PCR products) were fulfilled at the channel−probe line intersections and in a short time (minutes). The optimization of probe immobilization and sample hybridization are described in detail. The method successfully detected and discriminated between two 260 bp PCR products with a one-base-pair difference from closely related greenhouse plant fungal pathogens (B. cinerea and B. squamosa).

Keywords: Microfluidic microarray; DNA hybridization; fungal pathogen; single base-pair difference discrimination.

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