2013 추계 대한면역학회, 2013, 02, P-209─P-209

Highly Sensitive Detection of Bio-threat Pathogens by Oligonucleotide-linked Immunosorbent Assay using Gold-nanoparticle

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

Abstract

Detection and identification of pathogens from biological and environmental samples are a start line to treat infectious disease as well as to get epidemiological information. To meet with these demand, various pathogen detection methods have been developed, but the sensing approaches and conventional methods are not sufficient to identify the amount of infectious high-risk pathogens including Francisella tularensis (F. tularensis), Bacillus anthracis (B. anthracis), and Yersinia pestis (Y. pestis) due to their low infective dose. 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, and Y. pestis. GNP-OLISA uses two unique systems; 1) antibody-conjugated and DNA probe anchored 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 using 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 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.