Establishment and application of a loop‐mediated isothermal amplification−lateral flow dipstick (LAMP–LFD) method for detecting Clostridium piliforme

• Published in: Veterinary medicine and science 2024-01, Vol.10 (1), p.e1318-n/a
• Main Authors: Tao, Junhao, Yan, Huiqiong, Chen, Sisi, Du, Jiangtao, Zhou, Shasang, Guo, Honggang, Lu, Lingqun, Fang, Jie, Jin, Xiaoyin, Wang, Zhiyuan, Ying, Huazhong, Han, Wei, Dai, Fangwei
• Format: Article
• Language: English
• Subjects: Animal models; Animals; Biotin; China; Clostridiales; Clostridium; Clostridium piliforme; Cross-reactivity; diagnosis; Diarrhea; Disease; Enzyme-linked immunosorbent assay; Enzymes; Gene amplification; Humans; Infections; Laboratory animals; LAMP–LFD; Mice; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Amplification Techniques - veterinary; Original; Pathogens; rapid detection; RODENTS; rRNA; rRNA 23S; Salmonella; Sensitivity and Specificity; Serology; Specific pathogen free; Tyzzer; West Nile virus
• ISSN: 2053-1095; 2053-1095
• DOI: 10.1002/vms3.1318

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. In this study, we developed a method for the rapid detection of Clostridium piliforme. This method has good specificity, high sensitivity, simple operation and potential for basic promotion application.