Rapid and sensitive detection of Listeria monocytogenes by loop-mediated isothermal amplification
Meng-Jun Tang1, Sheng Zhou, Xiao-Yan Zhang
1Poultry Institute, Chinese Academy of Agricultural Sciences, Jiangsu, China. tangmengjun1980@163.com
Abstract:
Loop-mediated isothermal amplification (LAMP) was designed for detection of Listeria monocytogenes, which is an important food-borne kind of pathogenic bacteria causing human and animal disease. The primers set for the hlyA gene consist of six primers targeting eight regions on specific gene. The LAMP assay could be performed within 40 min at 65°C in a water bath. Amplification products were visualized by calcein and manganous ion and agarose gel electrophoresis. Sensitivity of the LAMP assay for detection of L. monocytogenes in pure cultures was 2.0 CFU per reaction. The LAMP assay was 100-fold higher sensitive than that of the conventional PCR assay. Taking this way, 60 chicken samples were investigated for L. monocytogenes. The accuracy of LAMP was shown to be 100% when compared to the "gold standard" culture-biotechnical, while the PCR assay failed to detect L. monocytogenes in two of the positive samples. It is shown that LAMP assay can be used as a sensitive, rapid, and simple detection tool for the detection of L. monocytogenes and will facilitate the surveillance for contamination of L. monocytogenes in food.
Insights
Loop-mediated isothermal amplification (LAMP) offers a rapid and sensitive method for detecting Listeria monocytogenes, a foodborne pathogen. This assay is highly accurate and outperforms conventional PCR for identifying contamination in food samples.
Area of Science:
- Microbiology
- Food Safety
- Molecular Diagnostics
Background:
- Listeria monocytogenes is a significant foodborne pathogen causing illness in humans and animals.
- Accurate and rapid detection methods are crucial for food safety surveillance.
- Existing methods like PCR may have limitations in sensitivity and speed.
Purpose of the Study:
- To develop and validate a Loop-mediated isothermal amplification (LAMP) assay for the detection of Listeria monocytogenes.
- To compare the sensitivity and accuracy of the LAMP assay against conventional PCR and culture-based methods.
- To assess the utility of LAMP for routine food sample analysis.
Main Methods:
- A novel primer set targeting the hlyA gene of Listeria monocytogenes was designed for LAMP.
- The LAMP assay was optimized for a 40-minute reaction time at 65°C.
- Amplification was visualized using calcein and manganous ion, with confirmation by agarose gel electrophoresis.
- Sensitivity was determined using pure cultures, and accuracy was evaluated using 60 chicken samples compared to culture-biotechnical methods.
Main Results:
- The LAMP assay demonstrated high sensitivity, detecting as few as 2.0 CFU/reaction of Listeria monocytogenes.
- The assay was 100-fold more sensitive than conventional PCR.
- In chicken samples, the LAMP assay achieved 100% accuracy, correctly identifying all positive samples, whereas PCR missed two positive cases.
- Amplification products were clearly visualized, facilitating straightforward interpretation.
Conclusions:
- Loop-mediated isothermal amplification (LAMP) provides a sensitive, rapid, and simple method for Listeria monocytogenes detection.
- The LAMP assay is a valuable tool for enhancing surveillance and ensuring the safety of food products.
- This method offers a significant improvement over conventional PCR for foodborne pathogen detection.
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