Multiplex PCR system for identifying the carnivore origins of faeces for an epidemiological study on Echinococcus multilocularis in Hokkaido, Japan

A multiplex PCR system was developed to identify the carnivore origins of faeces collected in Hokkaido, Japan, for epidemiological studies on Echinococcus multilocularis . Primers were designed against the D-loop region of mitochondrial DNA. Two separate primer mixtures (mix 1, specific forward primers to fox, raccoon dog and dog, and a universal reverse primer [prH]; and mix 2, specific forward primers to cat, raccoon and weasels and prH) were used so that the PCR products (160 bp, fox and cat; 240 bp, raccoon dog and raccoon; and 330 bp, dog and weasel) were distinguished by size. The multiplex PCR exhibited no cross-reactivity between carnivore species and did not amplify DNA from rodent prey. When 270 field-collected faeces were examined, 250 showed single PCR products belonging to specific target sizes, suggesting successful carnivore identification for 92.6% of samples. Taeniid eggs were detected in 11.1% of samples and coproantigen in 30.4%; whereas the prevalences of taeniid eggs and coproantigen were 12.9% and 34.0% in fox faeces, and 0% and 26.3% in cat faeces, respectively. These results suggest that the prevalence in different target animals can be evaluated individually and precisely using multiplex PCR system.