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

Abstract

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. To establish a method with good specificity, high sensitivity and simple operation for the detection of C. piliforme. 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. 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. 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.