title: Multiplex PCR system for identifying the carnivore origins of faeces for an epidemiological study on Echinococcus multilocularis in Hokkaido, Japan.
abstract: 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.
authors.affiliation: Laboratory of Veterinary Parasitic Diseases, Department of Veterinary Sciences, Faculty of Agriculture, University of Miyazaki, Gakuen-Kihanadai Nishi, Miyazaki, Japan. nnonaka@cc.miyazaki-u.ac.jp
authors.forename: Nariaki
authors.initials: N
authors.lastname: Nonaka
authors.forename: Takafumi
authors.initials: T
authors.lastname: Sano
authors.forename: Takashi
authors.initials: T
authors.lastname: Inoue
authors.forename: Maria
authors.initials: M
authors.lastname: Teresa Armua
authors.forename: Daisuke
authors.initials: D
authors.lastname: Fukui
authors.forename: Ken
authors.initials: K
authors.lastname: Katakura
authors.forename: Yuzaburo
authors.initials: Y
authors.lastname: Oku
mesh_terms: D000818:Animals; D002335:Carnivora / Q000235:genetics; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D004196:Disease Outbreaks; D004443:Echinococcosis / Q000453:epidemiology*; D048210:Echinococcus multilocularis / Q000302:isolation & purification*; D005243:Feces / Q000737:chemistry*; D007564:Japan / Q000453:epidemiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial