Using PCR amplification to increase the confidence level of Salmonella typhimurium DNA microarray chip hybridization.
In order to design and validate a method to identify virulence genes of Salmonella typhimurium using DNA microarray, a protocol was developed to label the isolated bacterial DNA directly and to use PCR amplification of limited numbers of genes to validate the hybridization signals. Therefore, a DNA microarray chip of 71 virulence genes of S. typhimurium was developed and evaluated using 10 isolates. Each gene was represented by 65bp oligonucleotide probes (oligoprobes) and immobilized on the surface of chemically modified slides. Whole DNA genomes were digested with Hinf1 and Sau3AI, labeled with a fluorescent tag of Cy3 and then hybridized. The presence of virulence genes in 10 strains of S. typhimurium was established by measuring a fluorescent signal above the background noise of the chip. PCR amplification of 10 genes (orgA, ORF319, ttrB, rmbA, misL, spi4F, spi4H, spi4N, rRNA, and purR) of S. typhimurium was used as a standard to verify the confidence level of the DNA microarray chip. In conclusion, using PCR amplification to increase the confidence level of the microarray hybridization data was successful.
HFS-517, Division of Microbiological Studies, Food and Drug Administration, Center for Food Safety and Applied Nutrition, 5100 Paint Branch Parkway, College Park, MD 20740-3855, USA.
Shannon
S
Courtney
Miriam E
ME
Mossoba
Thomas S
TS
Hammack
Christine
C
Keys
Sufian F
SF
Al-Khaldi
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012486:Salmonella typhimurium / Q000145:classification / Q000235:genetics*; D014774:Virulence / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S