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Published on: September 17, 2017
Magnetic nanoparticle-enhanced PCR for the detection and identification of Staphylococcus aureus and Salmonella
Dimitra Houhoula1, Joseph Papaparaskevas2, Katerina Zatsou1
1Department of Food Technology, Technological Educational Institution of Athens, 12210, Athens, Greece.
Abstract:
This paper evaluated magnetic nanoparticle-enhanced PCR for the detection and identification of Staphylococcus aureus and Salmonella enteritidis. Two different types of magnetic nanoparticles designated MPIO (iron concentration 2.5 mg/ml, size 1 µm) and NP (iron concentration 8.7 mg/ml, size 60 nm), both conjugated with S. aureus or S. enteritidis antibodies were evaluated as an enrichment procedure for PCR-detection of the pathogens in Trypticase Soy Broth, milk, blood and meat broth. Bacterial suspensions (1.5x108 cfu/ml) were prepared and serial diluted 10-1. The MPIO and NP nanoparticles were added, followed by incubation for 1 hour at room temperature, magnetic separation of the pellet, DNA extraction and PCR, targeting the femA and invA sequences. The nanoparticle-free and the NP-supplemented dilutions were positive down to the 1.5x102 cfu/ml concentration for both bacteria. The MPIO-supplemented dilutions were positive down to approx. 2x100 cfu/ml concentration, respectively. Bacteria-free TSB was negative by PCR. MPIO nanoparticles (size 1 µm) enhanced the detection of S. aureus and S. enteritidis by PCR, whilst NP nanoparticles (size 60 nm) did not, thus indicating that the size of the magnetic nanoparticles play a significant role in the enrichment procedure.
Insights
Magnetic nanoparticles significantly improved PCR detection of Staphylococcus aureus and Salmonella enteritidis. Larger MPIO nanoparticles enhanced pathogen identification, while smaller NP nanoparticles did not, highlighting the importance of nanoparticle size.
Area of Science:
- Biotechnology
- Molecular Biology
- Food Safety
Background:
- Accurate detection of foodborne pathogens like Staphylococcus aureus and Salmonella enteritidis is crucial for public health.
- Traditional PCR methods can be limited by low pathogen concentrations in complex matrices.
- Magnetic nanoparticles offer potential for enhancing pathogen capture and concentration prior to molecular detection.
Purpose of the Study:
- To evaluate the efficacy of magnetic nanoparticle-enhanced PCR for detecting Staphylococcus aureus and Salmonella enteritidis.
- To compare the performance of two distinct magnetic nanoparticles (MPIO and NP) in pathogen enrichment.
- To determine the influence of magnetic nanoparticle size on PCR detection sensitivity.
Main Methods:
- Two types of magnetic nanoparticles, MPIO (1 µm) and NP (60 nm), conjugated with specific antibodies, were used for pathogen enrichment.
- Enrichment was performed in various broths (Trypticase Soy Broth, milk, blood, meat broth) followed by magnetic separation.
- DNA was extracted from enriched samples, and PCR was performed targeting femA and invA genes for pathogen identification.
Main Results:
- MPIO nanoparticles (1 µm) significantly enhanced PCR detection sensitivity, enabling identification down to approximately 2x100 CFU/ml.
- NP nanoparticles (60 nm) did not improve detection sensitivity beyond standard PCR, with positive results down to 1.5x102 CFU/ml.
- Bacteria-free controls confirmed the specificity of the PCR assay and the absence of contamination.
Conclusions:
- Magnetic nanoparticle-enhanced PCR is a viable method for sensitive detection of Staphylococcus aureus and Salmonella enteritidis.
- The size of magnetic nanoparticles is a critical factor influencing their effectiveness in pathogen enrichment for PCR.
- Larger MPIO nanoparticles demonstrate superior performance for enhancing the detection limits of foodborne pathogens.
