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KMID : 0364820070430030179
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Korean Journal of Microbiology
2007 Volume.43 No. 3 p.179 ~ p.185
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Detection of Ralstonia solanacearum with Nested PCR and DNA Enzyme-Linked Immunosorbent Assay
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Ko Young-Jin
Cho Hong-Bum
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Abstract
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In this study, we used the method of guanidin isothiocyanate and boiling with Chelex-100 resin to extract genomic DNA of Ralstonia solanacearum from soil. It is more efficient than general protocols to remove inhibitory compounds in soil and R. solanacearum own. Then, we applied polymerase chain reaction and DNA enzyme-linked immunosorbent assay (ELISA) to identify and detect pathogen. The fliC gene of R. solanacearum was selected for specific detection of pathogen and primer sets were designed. Among the primer sets, two specific and sensitive primer sets, RsolfliC (forward; 5-GAACGCCAACGGTGCGAACT-3 and reverse; 5-GGCGGCCTTCAGGGAGGTC-3, designed by J. Schonfeld et al.) and RS_247 (forward; 5-GGCGGTCTGTCGGCRG-3 and reverse; 5-CGGTCGCGTTGGCAAC-3 designed by this study), were designed to perform nested PCR. Nested PCR primer was labeled with biotin for hybridization between nested PCR product and probe to analyze with DNA ELISA.
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KEYWORD
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DNA enzyme immunoassay, Ralstonia solanacearum
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