Specific identification and detection of Pantoea stewartii subsp. stewartii using a membrane-based multi-gene oligonucleotide array

2015-11-18T10:00:27Z (GMT) by James T. Tambong
<div><p></p><p>The Stewart’s wilt pathogen (<i>Pantoea stewartii</i> subsp. <i>stewartii</i>; Pss) of sweet corn is classified as a quarantine bacterium, requiring certification of grain shipments made to over 60 countries. This study reports the development and validation of the first multi-gene array for accurate and specific differentiation of Pss from <i>Pantoea stewartii</i> subsp. <i>indologenes</i> (Psi) and other <i>Pantoea</i> species using DNA, or RNA after generation of cDNA. The technique consisted of multiplex (16S rRNA, <i>leu</i>S, <i>gyr</i>B, <i>rpo</i>B, <i>cps</i>D) PCR amplifications using universal and specific primers in a single reaction, followed by the hybridization of the digoxigenin-labelled amplicons to 22 specific oligonucleotide probes (19- to 24-mers) immobilized on a nylon membrane. The sensitivity of the array (10 fg of DNA) compared favourably with TaqMan real-time PCR. The specificity and reliability of the array was tested on 65 bacterial strains consisting of <i>Pantoea</i> species, closely and distantly related bacterial genera. Specific detection of Pss in infected corn leaves and seed homogenates was also validated using growth chamber-inoculated plants. The reported multi-gene DNA array could be a reliable tool for routine and specific detection of Pss.</p></div>