2013 추계 화생방방어학회, 2013, 02, 35─35

Sensitive and Selective Identification of Bio-terror Agents by Oligonucleotide-linked immunosorbent Assay using Gold-nanoparticle

Sang-Hwan Seo, Dae-Ro Ahn, Ki-Cheol Han, Gi-Eun Rhie, Kee-Jong Hong

Abstract

Detection and identification of bio-terror agents including Francisella tularensis (F. tularensis), Bacillus anthracis (B. anthracis), and Yersinia pestis (Y. pestis) from biological and environmental samples are a start line to reduce risk of public health from these high-risk pathogens. Therefore, various pathogen detection methods have been developed, but the developed sensing approaches and conventional methods are not sufficient to identify the amount of infectious high-risk pathogens due to their low infective dose and high detection limit of the methods. In this study, we modified enzyme-linked immunosorbent assay (ELISA) using gold-nanoparticle (GNP) and oligonucleotide named as GNP-oligonucleotide-linked immunosorvent assay (OLISA) and adapted this sensing system to detect F. tularensis, B. anthracis spore and Y. pestis. GNP-OLISA uses two unique systems; 1) antibody and DNA probe-conjugated GNP as a signal generator and 2) RNA probe appended with fluorophore and quencher at both ends as a signal amplifier. Using GNP-OLISA, we could detect below 10 CFU of F. tularensis, 90 CFU of B. anthracis spore, and 50 CFU of Y. pestis from 100uL of PBS-diluted samples, which detection sensitivities are 21, 65, and 11 times higher than the results of corresponding ELISA, respectively. In addition, the calculated limit of detection values of GNP-OLISA using 100uL of rabbit serum-spiked F. tularensis, 1% skim-milk spiked B. anthracis spore, and rabbit serum-spiked Y. pestis sample were 29.63 ± 4.54 CFU/mL, 986.21 ± 146.58 CFU/mL, and 412.68 ± 177.48 CFU/mL, respectively, suggesting that detection sensitivities of GNP-OLISA for biological and food sample are not decreased. These results suggest that GNP-OLISA can be used as a sensitive detection method to overcome the detection limit of conventional method for high-risk pathogens.

• 본 연구는 질병관리본부 연구개발과제(과제번호 2013-NG45003-00) 연구비를 지원받아 수행되었습니다.
• This research was supported by a fund(code 2013-NG45003-00) by Research of Korea Centers for Disease Control and Prevention.