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Updated: May 5, 2026
On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System
Published on: February 23, 2018
Pathogen detection by core-shell type aptamer-magnetic preconcentration coupled to real-time PCR
V Cengiz Ozalp1, Gulay Bayramoglu2, Murat Kavruk3
1School of Medicine, Istanbul Kemerburgaz University, 34217 Istanbul, Turkey.
Abstract:
The presence of pathogenic bacteria is a major health risk factor in food samples and the commercial food supply chain is susceptible to bacterial contamination. Thus, rapid and sensitive identification methods are in demand for the food industry. Quantitative polymerase chain reaction (PCR) is one of the reliable specific methods with reasonably fast assay times. However, many constituents in food samples interfere with PCR, resulting in false results and thus hindering the usability of the method. Therefore, we aimed to develop an aptamer-based magnetic separation system as a sample preparation method for subsequent identification and quantification of the contaminant bacteria by real-time PCR. To achieve this goal, magnetic beads were prepared via suspension polymerization and grafted with glycidylmethacrylate (GMA) brushes that were modified into high quantities of amino groups. The magnetic beads were decorated with two different aptamer sequences binding specifically to Escherichia coli or Salmonella typhimurium. The results showed that even 1.0% milk inhibited PCR, but our magnetic affinity system capture of bacteria from 100% milk samples allowed accurate determination of bacterial contamination at less than 2.0 h with limit of detection around 100 CFU/mL for both bacteria in spiked-milk samples.
Insights
A new magnetic separation system using aptamers effectively isolates bacteria from food samples, enabling accurate detection via quantitative polymerase chain reaction (PCR) even in the presence of inhibitors like milk.
Area of Science:
- Food safety
- Molecular biology
- Biotechnology
Background:
- Bacterial contamination in food poses significant health risks.
- Current detection methods like quantitative polymerase chain reaction (PCR) are hindered by food matrix inhibitors.
- Rapid and sensitive bacterial identification is crucial for the food industry.
Purpose of the Study:
- To develop an aptamer-based magnetic separation technique for sample preparation.
- To enable accurate detection of contaminant bacteria using real-time PCR.
- To overcome PCR inhibition issues caused by food components.
Main Methods:
- Magnetic beads were synthesized and functionalized with amino groups.
- Aptamers specific to Escherichia coli and Salmonella typhimurium were immobilized on magnetic beads.
- The aptamer-magnetic bead system was used to capture bacteria from milk samples.
- Captured bacteria were quantified using real-time PCR.
Main Results:
- The magnetic separation system effectively captured bacteria from 100% milk samples.
- Accurate bacterial determination was achieved in under 2.0 hours.
- The limit of detection was approximately 100 CFU/mL for both E. coli and S. typhimurium.
- PCR inhibition by milk was overcome by the sample preparation method.
Conclusions:
- Aptamer-functionalized magnetic beads provide an efficient sample preparation method for bacterial detection in food.
- This system enhances the sensitivity and accuracy of quantitative PCR for food safety applications.
- The developed method offers a rapid and reliable solution for identifying bacterial contamination in complex food matrices.
