Related Experiment Video
Updated: Jul 11, 2025
Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation
Published on: May 20, 2020
Establishment and application of a loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for
Junhao Tao1,2, Huiqiong Yan1,2, Sisi Chen1,2
1Center of Laboratory Animal, Hangzhou Medical College, Hangzhou, China.
Background:
Clostridium piliforme (causative agent of Tyzzer disease) infects various animals, including primates, and hence a threat to animal and human health worldwide. At present, it is detected using traditional methods, such as path morphology, polymerase chain reaction and enzyme-linked immunosorbent assay. Therefore, it is necessary to develop convenient, efficient visual molecular biological methods for detecting C. piliforme.
Objectives:
To establish a method with good specificity, high sensitivity and simple operation for the detection of C. piliforme.
Methods:
In this study, we designed internal and external primers based on the conserved 23S rRNA region of C. piliforme to develop a biotin-labelled diarrhoea-suffered loop-mediated isothermal amplification (LAMP) system for detecting of C. piliforme and assessed the specificity, sensitivity and repeatability of the LAMP system.
Results:
The LAMP system did not exhibit cross-reactivity with 24 other common pathogenic species, indicating that it had good specificity. The minimum concentration of sensitivity was 1 × 10-7 ng/μL. Mouse models (Meriones unguiculatus) of Tyzzer disease were established and a LAMP-lateral flow dipstick (LAMP-LFD) was developed for detecting C. piliforme. The detection rate of C. piliforme was 5.08% in clean-grade animals and 9.96% in specific-pathogen-free-grade animals from Jiangsu, Zhejiang and Shanghai. In addition, the detection rates of C. piliforme were 10.1%, 8.6% and 20%, in animals from Hangzhou, Wenzhou and Shaoxing, respectively. The detection rate of C. piliforme was higher in experimental animals used in schools than in those used in companies and research institutes.
Conclusions:
The LAMP-LFD method established in this study can be used to detect C. piliforme in animals handled in laboratory facilities of universities, pharmaceutical enterprises and research and development institutions.
Insights
A new loop-mediated isothermal amplification (LAMP) method offers a specific and sensitive way to detect Clostridium piliforme, the cause of Tyzzer disease. This visual detection system aids in identifying the pathogen in laboratory animals, improving animal and human health surveillance.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Clostridium piliforme causes Tyzzer disease, a global threat to animal and human health.
- Current detection methods for C. piliforme lack convenience and efficiency.
- There is a need for rapid, visual molecular diagnostic tools for C. piliforme.
Purpose of the Study:
- To develop a highly specific, sensitive, and user-friendly method for detecting C. piliforme.
- To establish a visual molecular diagnostic tool for C. piliforme detection.
Main Methods:
- Designed primers targeting the conserved 23S rRNA region of C. piliforme.
- Developed a biotin-labelled loop-mediated isothermal amplification (LAMP) system.
- Assessed specificity, sensitivity, and repeatability using various pathogenic species and concentrations.
- Developed a LAMP-lateral flow dipstick (LAMP-LFD) assay and tested it on animal samples.
Main Results:
- The LAMP system demonstrated high specificity, with no cross-reactivity against 24 other pathogens.
- Achieved a minimum detection sensitivity of 1 × 10⁻⁷ ng/μL.
- Detected C. piliforme in laboratory animals, with varying prevalence rates across different regions and types of facilities.
- The LAMP-LFD assay successfully identified C. piliforme in tested animal populations.
Conclusions:
- The developed LAMP-LFD method provides a convenient, efficient, and visual means for detecting C. piliforme.
- This assay is suitable for use in laboratory animal facilities within academic, pharmaceutical, and research institutions.
- The method contributes to improved surveillance and control of Tyzzer disease in animal populations.
