Multiple detection of food-borne pathogenic bacteria using a novel 16S rDNA-based oligonucleotide signature chip.
There have been many attempts to develop sensitive and accurate techniques for the detection and diagnosis of pathogenic bacteria using nucleic acid-based technology. To achieve efficient multiple detection of seven selected food-borne pathogens, we assessed the respective 16S rDNA pathogen specific sequences using an oligonucleotide-based signature array. Strategic optimal design of specific capture probes was achieved by using the characteristic first variable region. To assess the specificity of this pathogen detection system, we employed a two-step experimental strategy. Under conditions established through experiments with chemically synthesized model targets comprising both conserved and variable regions of 16S rDNA, we confirmed the validity of this system using real 16S rDNA targets. Detection with real targets was successfully performed using our system, and better specificity was obtained compared to experiments with model targets. Moreover, the subtypes of Vibrio pathogens were successfully classified. We developed a two-dimensional visualization plot tool for positive control and specific spots, which allowed facile and minute differentiation between spot intensities. Repeated array formats were employed to ensure experimental uniformity, and included the statistical p-value criterion for pathogen discrimination. The present results thus indicate that our novel oligonucleotide-based signature chip detection system can be employed for the effective detection of multiple pathogens.
Eom
Ho Seop
HS
Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.
Hwang
Byeong Hee
BH
Kim
Duk-Hee
DH
Lee
In-Beum
IB
Kim
Young Hee
YH
Cha
Hyung Joon
HJ
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005504:Food Analysis / Q000295:instrumentation / Q000379:methods; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S