title: Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay.
abstract: BACKGROUND
  Infections of Echinococcus granulosus sensu stricto (s.s), E. multilocularis and E. shiquicus are commonly found co-endemic on the Qinghai-Tibet plateau, China, and an efficient tool is needed to facilitate the detection of infected hosts and for species identification.
  
  
  RESULTS
  A single-tube multiplex PCR assay was established to differentiate the Echinococcus species responsible for infections in intermediate and definitive hosts. Primers specific for E. granulosus, E. multilocularis and E. shiquicus were designed based on sequences of the mitochondrial NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 5 (nad5) and cytochrome c oxidase subunit 1 (cox1) genes, respectively. This multiplex PCR accurately detected Echinococcus DNA without generating nonspecific reaction products. PCR products were of the expected sizes of 219 (nad1), 584 (nad5) and 471 (cox1) bp. Furthermore, the multiplex PCR enabled diagnosis of multiple infections using DNA of protoscoleces and copro-DNA extracted from fecal samples of canine hosts. Specificity of the multiplex PCR was 100% when evaluated using DNA isolated from other cestodes. Sensitivity thresholds were determined for DNA from protoscoleces and from worm eggs, and were calculated as 20 pg of DNA for E. granulosus and E. shiquicus, 10 pg of DNA for E. multilocularis, 2 eggs for E. granulosus, and 1 egg for E. multilocularis. Positive results with copro-DNA could be obtained at day 17 and day 26 after experimental infection of dogs with larval E. multilocularis and E. granulosus, respectively.
  
  
  CONCLUSIONS
  The multiplex PCR developed in this study is an efficient tool for discriminating E. granulosus, E. multilocularis and E. shiquicus from each other and from other taeniid cestodes. It can be used for the detection of canids infected with E. granulosus s.s. and E. multilocularis using feces collected from these definitive hosts. It can also be used for the identification of the Echinococcus metacestode larva in intermediate hosts, a stage that often cannot be identified to species on visual inspection.
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Key Laboratory of Zoonoses of Agriculture Ministry/Lanzhou Veterinary Research Institute, CAAS, Lanzhou, People's Republic of China.
authors.forename: Cong-Nuan
authors.initials: CN
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Key Laboratory of Zoonoses of Agriculture Ministry/Lanzhou Veterinary Research Institute, CAAS, Lanzhou, People's Republic of China.
authors.forename: Zhong-Zi
authors.initials: ZZ
authors.lastname: Lou
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Key Laboratory of Zoonoses of Agriculture Ministry/Lanzhou Veterinary Research Institute, CAAS, Lanzhou, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Key Laboratory of Zoonoses of Agriculture Ministry/Lanzhou Veterinary Research Institute, CAAS, Lanzhou, People's Republic of China.
authors.forename: Hong-Bin
authors.initials: HB
authors.lastname: Yan
authors.affiliation: School of Marine and Tropical Biology, James Cook University, Queensland, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Blair
authors.affiliation: Qinghai Academy of Animal Science and Veterinary Medicine, Xining, People's Republic of China.
authors.forename: Meng-Tong
authors.initials: MT
authors.lastname: Lei
authors.affiliation: Qinghai Academy of Animal Science and Veterinary Medicine, Xining, People's Republic of China.
authors.forename: Jin-Zhong
authors.initials: JZ
authors.lastname: Cai
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Key Laboratory of Zoonoses of Agriculture Ministry/Lanzhou Veterinary Research Institute, CAAS, Lanzhou, People's Republic of China.
authors.forename: Yan-Lei
authors.initials: YL
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Key Laboratory of Zoonoses of Agriculture Ministry/Lanzhou Veterinary Research Institute, CAAS, Lanzhou, People's Republic of China.
authors.forename: Jian-Qiu
authors.initials: JQ
authors.lastname: Li
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Key Laboratory of Zoonoses of Agriculture Ministry/Lanzhou Veterinary Research Institute, CAAS, Lanzhou, People's Republic of China.
authors.forename: Bao-Quan
authors.initials: BQ
authors.lastname: Fu
authors.affiliation: Molecular Parasitology Laboratory, Infectious Diseases Division, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, People's Republic of China.
authors.forename: Yu-Rong
authors.initials: YR
authors.lastname: Yang
authors.affiliation: Molecular Parasitology Laboratory, Infectious Diseases Division, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors.forename: Donald P
authors.initials: DP
authors.lastname: McManus
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Key Laboratory of Zoonoses of Agriculture Ministry/Lanzhou Veterinary Research Institute, CAAS, Lanzhou, People's Republic of China.
authors.forename: Wan-Zhong
authors.initials: WZ
authors.lastname: Jia
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics; D004285:Dogs; D004443:Echinococcosis / Q000175:diagnosis* / Q000469:parasitology* / Q000662:veterinary; D004446:Echinococcus / Q000145:classification* / Q000235:genetics*; D003576:Electron Transport Complex IV / Q000235:genetics; D005243:Feces / Q000469:parasitology; D006801:Humans; D051379:Mice; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009245:NADH Dehydrogenase / Q000235:genetics; D010274:Parasitology / Q000379:methods*; D012680:Sensitivity and Specificity; D018609:Tibet / Q000453:epidemiology
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D009245:NADH Dehydrogenase; D003576:Electron Transport Complex IV