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Updated: Mar 28, 2026
Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation
Published on: May 20, 2020
Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time
Yi Wang1, Yan Wang1, Lijuan Luo1
1State Key Laboratory of Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention Beijing, China.
Abstract:
Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples.
Insights
A new method, multiple endonuclease restriction real-time loop-mediated isothermal amplification (MERT-LAMP), simultaneously detects Shigella and Salmonella. This rapid, sensitive assay offers a valuable tool for food safety screening.
Area of Science:
- Molecular Biology
- Food Microbiology
- Diagnostic Technology
Background:
- Shigella and Salmonella are common foodborne pathogens causing human gastroenteritis.
- Existing detection methods can be time-consuming and require multiple steps.
- There is a need for rapid, simultaneous detection of these bacteria in food samples.
Purpose of the Study:
- To establish and validate a novel multiplex assay for simultaneous detection of Shigella and Salmonella.
- To develop a rapid, sensitive, and efficient diagnostic tool for food safety applications.
Main Methods:
- Development of multiple endonuclease restriction real-time loop-mediated isothermal amplification (MERT-LAMP) technology.
- Design of specific primers targeting the ipaH gene of Shigella spp. and the invA gene of Salmonella spp.
- Validation using genomic DNA from reference strains and artificially contaminated milk samples.
Main Results:
- Simultaneous detection of Shigella and Salmonella achieved in a single reaction within 12 minutes at 63°C.
- Distinct fluorescence curves allowed real-time differentiation of pathogens.
- MERT-LAMP demonstrated high analytical sensitivity (62.5-125 fg DNA/reaction) and low limits of detection in milk (5.8-6.4 CFU/vessel), outperforming qPCR and conventional PCR.
Conclusions:
- The MERT-LAMP methodology provides a rapid, sensitive, and efficient means for simultaneous screening of Shigella and Salmonella.
- This novel assay significantly reduces workload, materials, and reagents compared to conventional methods.
- MERT-LAMP is a valuable tool for the simultaneous detection of these foodborne pathogens in diverse sample types.
