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A sensitive method to detect Escherichia coli based on immunomagnetic separation and real-time PCR amplification of aptamers by Hye-Jin Lee; Byoung Chan Kim; Kyung-Woo Kim; Young Keun Kim; Jungbae Kim; Min-Kyu Oh is a Engineering article available to read on EtoBox.

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Aptamers, single-stranded nucleic acids, provide a unique opportunity as amplifiable molecules using polymerase chain reaction (PCR) as well as recognition molecules like antibodies. We report a highly sensitive detection of Escherichia coli by taking advantage of the aptamer amplification as well as the specific binding of aptamers onto E. coli. This unique approach consists of three steps. First, the target E. coli was captured by antibody-conjugated magnetic beads. Second, the RNA aptamers were bound onto the surface of captured E. coli in a sandwich way. Finally, the heat-released aptamers were amplified by using real-time reverse-transcriptase-PCR (RT-PCR). The aptamer amplification in this approach has enabled a sensitive detection of microorganisms, such as the detection of 10 E. coli in 1 ml sample. When compared to the amplification of nucleic acids extracted from the target microorganisms, this approach can not only prevent the loss of target nucleic acids during the sample preparation by obviating the necessity of cell lysis, but also provide an additional mechanism of signal amplification due to the binding of many aptamers to the surface of each E. coli. Detection of E

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Author

Hye-Jin Lee; Byoung Chan Kim; Kyung-Woo Kim; Young Keun Kim; Jungbae Kim; Min-Kyu Oh

Publisher

Elsevier Science; Elsevier ; Elsevier Ltd.; Elsevier BV (ISSN 0956-5663)

Published

2009

Language

EN

Field

Engineering (Physical Sciences)