Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays

Abstract

We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.

FIG. 1.

Probe specificity. Positive-control hybridization signals for A. hydrophila (AEHY), A. salmonicida subsp. salmonicida (AESA), E. ictaluri (EDIC), E. coli (ESCO), F. branchiophilum (FLBR), F. columnare (FLCO), F. psychrophilum (FLPS), M. chelonae (MYCH), M. fortuitum (MYFO), M. marinum (MYMA), R. salmoninarum (RESA), P. salmonis (PISA), S. aureus (STAU), S. iniae (STIN), T. maritimum (TEMA), V. salmoninarum (VASA), P. phosphoreum (PHPH), and Y. ruckeri type I (YERU) are shown. The lower right row of spots in each panel (not shown for MYMA or MYCH) is a biotinylated oligonucleotide control for detection chemistry.

FIG. 2.

Representative examples of microarray hybridization from no-template control PCRs. Little to no background is evident with 30 cycles or less, but considerable background is evident with 35 cycles of PCR.

FIG. 3.

Assay sensitivity. Data showing assay sensitivity for purified pathogen DNA alone (•) or for DNA with competing bacterial (○) or fish (▾) DNA. Competing DNA does not impact assay sensitivity (<150 copies; P < 0.05) using this protocol.