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Updated: May 6, 2026
Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
Published on: March 13, 2018
Rapid and simple detection of Listeria monocytogenes using real closed dumbbell-mediated isothermal amplification
Yanli Zhang1, Xinyao Wu2, Yeling Zhong3
1Department of Hematology, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China.
Introduction:
Listeria monocytogenes (L. monocytogenes) is a well-known widespread food-borne pathogen that poses a threat to public health. Suitable detection methods are needed to effectively control and prevent pathogenic L. monocytogenes infections.
Methods:
This study aimed to develop a novel closed dumbbell-mediated isothermal amplification (CDA)-based assay to achieve rapid and simple detection of L. monocytogenes. The newly developed CDA technology is capable of amplifying DNA targets with high sensitivity and specificity. The conserved hly gene of L. monocytogenes was taken as a target for the establishment of the CDA method. All primers were selected and evaluated by real-time fluorescence monitoring, and endpoint visual judgement was indicated by hydroxy naphthol blue (HNB).
Results:
The specificity and sensitivity of this CDA-based diagnostic system were determined after the evaluation of 560 batches of DNA samples. The detection limit of the L. monocytogenes O-CDA assay was 1 copy/μl using artificial samples. The results of real-time fluorescence-based O-CDA coupled with melting curves analysis showed that the method would achieve rapid and accurate diagnosis of L. monocytogenes, and can be employed as an alternative to qPCR in diagnostic practice. Moreover, the L. monocytogenes O-CDA method monitored by real-time fluorescence and endpoint hydroxy naphthol blue (HNB) based colorimetric assay displayed the same sensitivity, specificity, and accuracy, which would be helpful to realize onsite pathogen surveillance.
Discussion:
The real-time fluorescence plots and following melting curve analysis-based L. monocytogenes O-CDA were suitable for laboratory-based diagnosis. Considering the portable manipulation of the HNB-based colorimetric detection system, our results shed light on its potential application for on-site L. monocytogenes surveillance. The developed CDA-based methods are rapid, simple, reliable, and sensitive in several samples, showing potential to manage the task of L. monocytogenes monitoring more easily.
Insights
A new closed dumbbell-mediated isothermal amplification (CDA) assay rapidly and simply detects Listeria monocytogenes. This sensitive and specific method offers a viable alternative for pathogen surveillance, suitable for both lab and on-site applications.
Area of Science:
- Molecular Biology
- Food Safety
- Diagnostic Assays
Background:
- Listeria monocytogenes is a significant foodborne pathogen requiring effective detection methods.
- Current detection methods may lack the speed or simplicity needed for widespread surveillance.
- Rapid and accurate diagnostics are crucial for public health protection.
Purpose of the Study:
- To develop a novel closed dumbbell-mediated isothermal amplification (CDA) assay for Listeria monocytogenes detection.
- To establish a rapid, simple, sensitive, and specific diagnostic tool for L. monocytogenes.
- To evaluate the potential of CDA for both laboratory and on-site pathogen surveillance.
Main Methods:
- Developed a closed dumbbell-mediated isothermal amplification (CDA) assay targeting the hly gene of L. monocytogenes.
- Utilized real-time fluorescence monitoring and melting curve analysis for detection.
- Incorporated hydroxy naphthol blue (HNB) for endpoint visual colorimetric detection.
- Validated the assay using 560 DNA samples to assess specificity and sensitivity.
Main Results:
- The L. monocytogenes O-CDA assay demonstrated a detection limit of 1 copy/μl.
- Real-time fluorescence and melting curve analysis provided rapid and accurate diagnosis, comparable to qPCR.
- The HNB-based colorimetric assay showed equivalent sensitivity, specificity, and accuracy for on-site surveillance potential.
- The assay proved effective across various sample types.
Conclusions:
- The developed CDA-based assay is a rapid, simple, reliable, and sensitive method for L. monocytogenes detection.
- Real-time fluorescence CDA is suitable for laboratory diagnostics.
- HNB-based colorimetric CDA holds promise for on-site pathogen surveillance.
- These CDA methods offer an improved approach for managing L. monocytogenes monitoring.
