Methodologies towards the development of an oligonucleotide microarray for determination of Salmonella serotypes.
A DNA-based microarray designed to detect somatic (O) and flagellar (H) antigens present in the five most commonly isolated Salmonella serovars within Canada was developed as an alternative to the traditional Kauffmann-White serotyping scheme currently used to serotype salmonellae. Short oligonucleotide probes were designed based on publicly available sequence data of selected genes responsible for O and H antigen biosynthesis. These targets included: antigen-specific sequences within the flagella (H) antigen phase 1 (fliC) and phase 2 (fljB) genes and somatic (O) antigen biosynthesis genes within the rfb cluster (Groups B--rfbJ, C1--wbaA, C2--rfbJ, D1--rfbS). A prototype microarray with 117 O and H antigen-specific probes and controls was used to assess probe performance against two pools of gene target PCR amplicons. A set of 31 of these antigen-specific probes (8 O and 23 H) with high specific signal and low non-specific signal were selected based on t-test (p-value <0.01) and log(2) ratio distribution analysis to create a prototype microarray. The microarray was tested against 16 Salmonella strains of known serotype. Based on the strains tested in this study, these probes successfully identified and differentiated 11 of the 12 antigens targeted. The prototype DNA-based typing microarray described here has the potential to be an automated alternative to the traditional antigen-antibody serotyping scheme currently used for Salmonella.
Yoshida
Catherine
C
Public Health Agency of Canada, Laboratory for Foodborne Zoonoses, 110 Stone Road West, Guelph, ON, Canada N1G 3W4. catherine_yoshida@phac-aspc.gc.ca
Franklin
Kristyn
K
Konczy
Paulina
P
McQuiston
John R
JR
Fields
Patricia I
PI
Nash
John H
JH
Taboada
Ed N
EN
Rahn
Kris
K
D000942:Antigens, Bacterial / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000235:genetics; D005408:Flagellin / Q000235:genetics; D019081:O Antigens / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D012475:Salmonella / Q000145:classification* / Q000235:genetics*; D012680:Sensitivity and Specificity
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C094427:FljB protein, Salmonella; C022863:H antigen; D019081:O Antigens; C079397:rfb protein, Bacteria; D005408:Flagellin