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Updated: May 4, 2026
Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB
Published on: August 6, 2011
A modified molecular beacons-based multiplex real-time PCR assay for simultaneous detection of eight foodborne
Qinghua Hu1, Dongyue Lyu, Xiaolu Shi
11 Shenzhen Centre for Disease Control and Prevention , Shenzhen, Guangdong, China .
Abstract:
Foodborne disease outbreaks are often caused by one of the major pathogens. Early identification of the causal pathogen is crucial for disease control and prevention. We describe a real-time polymerase chain reaction (rtPCR) assay that can identify, in a single reaction, up to eight common foodborne bacterial pathogens, including Salmonella enterica subsp. enterica, Listeria monocytogenes, Escherichia coli O157, Vibrio parahaemolyticus, V. vulnificus, Campylobacter jejuni, Enterobacter sakazakii, and Shigella spp. This multiplex rtPCR assay takes advantage of modified molecular beacons and the multicolor combinational probe coding strategy to discriminate each pathogen and the homo-tag assisted non-dimer (HAND) system to prevent dimer formation. The detection limits of the assay ranged from 1.3×10(3) colony-forming units (CFU)/g stool (L. monocytogenes) to 1.6×10(4) CFU/g stool (Shigella spp.). The target genes were 100% specific as assessed on 986 reference strains covering 41 species since no cross-reactions were observed. The assay was applied to the detection of foodborne pathogens in 11,167 clinical samples and the results were compared with culture methods for further validation. The sensitivity and specificity of the rtPCR were 100% and 99%, respectively. When performed in a 96-well rtPCR system, more than 90 samples could be analyzed within 3 h. Given the high accuracy, sensitivity, specificity, and short turn-around time, the established assay could be used for the rapid and reliable identification of the causative pathogens responsible for a certain foodborne disease outbreak and rapid screening of these major foodborne pathogens in laboratory-based surveillance of outpatient clinical samples or even food samples.
Insights
A new multiplex real-time polymerase chain reaction (rtPCR) assay rapidly identifies up to eight common foodborne pathogens in a single reaction. This highly accurate assay offers a fast and reliable method for outbreak investigation and surveillance.
Area of Science:
- Microbiology
- Molecular Biology
- Public Health
Background:
- Foodborne disease outbreaks pose significant public health risks.
- Early and accurate identification of causative pathogens is critical for effective control and prevention strategies.
Purpose of the Study:
- To develop and validate a multiplex real-time polymerase chain reaction (rtPCR) assay for the simultaneous detection of eight common foodborne bacterial pathogens.
- To assess the assay's performance in clinical samples compared to traditional culture methods.
Main Methods:
- A multiplex rtPCR assay utilizing modified molecular beacons and multicolor combinational probe coding.
- Incorporation of the homo-tag assisted non-dimer (HAND) system to prevent dimer formation.
- Validation using 986 reference strains and 11,167 clinical samples, with comparison to culture-based methods.
Main Results:
- The assay detected up to eight common foodborne pathogens, including Salmonella, Listeria, E. coli O157, Vibrio, Campylobacter, Enterobacter, and Shigella.
- Detection limits ranged from 1.3×10^3 CFU/g to 1.6×10^4 CFU/g.
- 100% specificity was observed against 986 reference strains. In clinical samples, the assay demonstrated 100% sensitivity and 99% specificity.
- Over 90 samples could be analyzed within 3 hours using a 96-well rtPCR system.
Conclusions:
- The developed multiplex rtPCR assay provides a rapid, accurate, and sensitive method for identifying major foodborne pathogens.
- This assay is suitable for timely identification of pathogens in foodborne disease outbreaks and for laboratory-based surveillance of clinical and food samples.
