Related Experiment Video
Updated: Feb 7, 2026
Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System
Published on: November 21, 2023
A multiplex PCR amplification strategy coupled with microchip electrophoresis for simultaneous and sensitive
Luqi Zhu1, Yating Zhang1, Pingang He1
1School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, PR China.
Abstract:
Foodborne bacteria are some of the most important human pathogens and can cause many diseases. In this study, multiplex PCR amplification combined with microchip electrophoresis (MCE) was studied to simultaneously and sensitively detect Staphylococcus aureus, Proteus mirabilis, and Enterobacter sakazakii. In order to simultaneously and accurately detect the aim bacteria, three pairs of primers were specially designed for the multiplex PCR amplification of the target genes of three bacteria, which were the specific genes corresponding to these bacteria respectively. After the DNA fragments of three bacteria were simultaneously extracted, the multiplex PCR amplification was performed by adding the three pairs of specific primers in the mixed DNA fragments solution. The multiplex PCR products of the three food-borne pathogens were analyzed by MCE and the limits of detection of target DNA fragments were 1.2-2.2ngμL-1, (S/N=3). The limits of detection of the aim bacteria were calculated as 53CFUmL-1 for Enterobacter sakazakii, 32CFUmL-1 for Proteus mirabilis, 28CFUmL-1 for Staphylococcus aureus, respectively. Satisfactory results were obtained when this method was applied to detect the three foodborne bacteria in milk samples. The experimental results show that this method has the advantages of quickness, less sample consumption, high selectivity and high sensitivity.
Insights
This study introduces a rapid multiplex PCR and microchip electrophoresis method for detecting foodborne pathogens like Staphylococcus aureus, Proteus mirabilis, and Enterobacter sakazakii simultaneously. The technique offers high sensitivity and selectivity, proving effective in milk sample analysis.
Area of Science:
- Food microbiology
- Molecular diagnostics
- Pathogen detection
Background:
- Foodborne bacterial infections pose significant public health risks.
- Accurate and rapid detection of multiple pathogens is crucial for food safety.
- Existing methods may lack the sensitivity or speed for comprehensive screening.
Purpose of the Study:
- To develop a simultaneous detection method for key foodborne pathogens: Staphylococcus aureus, Proteus mirabilis, and Enterobacter sakazakii.
- To combine multiplex PCR amplification with microchip electrophoresis (MCE) for enhanced sensitivity and specificity.
- To validate the method's efficacy in detecting these bacteria in food matrices, specifically milk.
Main Methods:
- Design of specific primer pairs for multiplex PCR targeting unique genes of S. aureus, P. mirabilis, and E. sakazakii.
- Simultaneous DNA extraction from bacterial samples.
- Multiplex PCR amplification followed by analysis using microchip electrophoresis (MCE).
- Determination of limits of detection for DNA fragments and bacterial concentrations.
Main Results:
- Successful simultaneous amplification and detection of target genes from the three bacteria.
- Achieved limits of detection for DNA fragments between 1.2-2.2 ng/µL (S/N=3).
- Determined bacterial limits of detection: 28 CFU/mL for S. aureus, 32 CFU/mL for P. mirabilis, and 53 CFU/mL for E. sakazakii.
- Demonstrated satisfactory performance in detecting these pathogens in spiked milk samples.
Conclusions:
- The developed multiplex PCR combined with MCE is a highly sensitive and selective method for simultaneous detection of S. aureus, P. mirabilis, and E. sakazakii.
- This approach offers advantages such as speed, minimal sample consumption, and high accuracy.
- The method is suitable for routine application in food safety testing, particularly for milk products.
Related Concept Videos
01:32PCR
01:20Spin–Spin Coupling: Two-Bond Coupling (Geminal Coupling)
The central atom need not be NMR-active because its electrons are affected by the electron polarization of the spin-active atoms. However, spin information is transmitted less effectively than in one-bond coupling, and 2J values are usually weaker than 1J values. The energy of...
01:22Spin–Spin Coupling: Three-Bond Coupling (Vicinal Coupling)
The extent of coupling depends on the C‑C bond length, the two H‑C‑C angles, any electron-withdrawing substituents, and the dihedral angle between the involved orbitals. The...
01:20Electrophoresis: Overview
There...
01:21G-protein Coupled Receptors
01:17Spin–Spin Coupling: One-Bond Coupling