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

PLoS Negl Trop Dis. 2015 Sep 22;9(9):e0004084. doi: 10.1371/journal.pntd.0004084. eCollection 2015 Sep.

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.

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 (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/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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA Primers / genetics
  • DNA, Mitochondrial / genetics
  • Dogs
  • Echinococcosis / diagnosis*
  • Echinococcosis / parasitology*
  • Echinococcosis / veterinary
  • Echinococcus / classification*
  • Echinococcus / genetics*
  • Electron Transport Complex IV / genetics
  • Feces / parasitology
  • Humans
  • Mice
  • Molecular Diagnostic Techniques / methods*
  • Multiplex Polymerase Chain Reaction / methods*
  • NADH Dehydrogenase / genetics
  • Parasitology / methods*
  • Sensitivity and Specificity
  • Tibet / epidemiology

Substances

  • DNA Primers
  • DNA, Mitochondrial
  • NADH Dehydrogenase
  • Electron Transport Complex IV

Grants and funding

This study was supported by the following: WZJ: Gansu Provincial Key Science and Technology Projects (Grant No. 1203NKDA039, http://www.gsstc.gov.cn/), ZZL: the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201303037, http://www.moa.gov.cn), WZJ: the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 200903036-07, http://www.moa.gov.cn), WZJ: the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006, http://www.gsstc.gov.cn/), YRY: Natural Science Foundation of China (Grant No. 30960339, http://www.nsfc.gov.cn/), YRY: National Health and Medical Research Council (NHMRC) Project (Grant No. APP-1009539, http://www.nhmrc.gov.au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.