A sensitive method to detect Escherichia coli based on immunomagnetic separation and real-time PCR amplification of aptamers

Hye Jin Lee, Byoung Chan Kim, Kyung Woo Kim, Young-geun Kim, Jungbae Kim, Min-Kyu Oh

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

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. coli in this approach showed a wide dynamic range from 101 to 107 E. coli per ml, which can be explained by the exponential amplification of aptamers. This report has demonstrated, for the first time, the effective use of aptamer amplification in the development of sensitive microorganism detection. It is anticipated that the present approach will be easily expanded and employed in various types of microorganism detection.

Original languageEnglish
Pages (from-to)3550-3555
Number of pages6
JournalBiosensors and Bioelectronics
Volume24
Issue number12
DOIs
Publication statusPublished - 2009 Aug 15

Keywords

  • Aptamers
  • Immunomagnetic separation
  • Microorganism detection
  • Real-time RT-PCR

ASJC Scopus subject areas

  • Biophysics
  • Biomedical Engineering
  • Biotechnology
  • Electrochemistry

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