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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.    Methodology/Principal Findings  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 ( nad 1), NADH dehydrogenase subunit 5 ( nad 5) and cytochrome c oxidase subunit 1 ( cox 1) genes, respectively. This multiplex PCR accurately detected  Echinococcus  DNA without generating nonspecific reaction products. PCR products were of the expected sizes of 219 ( nad 1), 584 ( nad 5) and 471 ( cox 1) 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/Significance  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.

Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay