One step Multiplex PCR for detection and genotyping of Innovation for the Management of Echinococcosis Besançon, 2014 One step Multiplex PCR for detection and genotyping of E. granulosus complex Ghalia BOUBAKER, Bruno GOTTSTEIN, Markus SPILIOTIS Institute of Parasitology, Vetsuisse Faculty Berne, Langgass-Str. 122, CH-3012 Berne, Switzerland.

I- Introduction 10 genotypes E. granulosus complex Recent taxonomy One step Multiplex PCR for detection and genotyping of E. granulosus complex I- Introduction E. granulosus is characterized by a large intraspecific genetic variability 10 genotypes E. granulosus complex (Nakao et al., 2010) Recent taxonomy Common sheep strain (G1) E. granulosus s. s. (G1/2/3) Tasmanian sheep strain (G2) Buffalo strain (G3) Horse strain (G4) E. equinus (G4) Cattle strain (G5) E. ortleppi (G5) Camel strain (G6) Pig strain (G7) E. canadensis (G6-10) Cervid strain (G8/G10)

Two procedures are available for genotyping of E. granulosus: One step Multiplex PCR for detection and genotyping of E. granulosus complex I- Introduction Molecular genotyping of E. granulosus strains is important to understand their temporal and spatial distributions Two procedures are available for genotyping of E. granulosus: Two steps systems: Detection (PCR, cox and ndI) and genotyping (Sequencing, RFLP, Fingerprinting, SSCP). Multistep pure PCR based systems: Three PCR consecultive to differentiate between E. granulosus s.s. (G1), E. canadensis (G6/G7) and E. ortleppi (G5) (Dinkel et al., 2008).

Primer design: nuclear and mitochondrial gene markers One step Multiplex PCR for detection and genotyping of E. granulosus complex II- Multiplex-PCR Primer design: nuclear and mitochondrial gene markers

The optimized protocol of the multiplex-PCR: One step Multiplex PCR for detection and genotyping of E. granulosus complex II- Multiplex-PCR The optimized protocol of the multiplex-PCR: eleven sets of primers/ 25 cycles/ 5ng DNA E. granulosus s.s. (G1) E. equinus (G4) E. Equinus (G4) E. ortleppi (G5) E. canadensis (G6/7) E. canadensis (G8/10) E. multilocularis E. vogeli Echinococcus

Validation of the multiplex-PCR One step Multiplex PCR for detection and genotyping of E. granulosus complex II- Multiplex-PCR Validation of the multiplex-PCR DNA extracted from metacestode larval stage

The multiplex-PCR is able to detect co-infection cases One step Multiplex PCR for detection and genotyping of E. granulosus complex II- Multiplex-PCR The multiplex-PCR is able to detect co-infection cases Experimental DNA mixing test E. granulosus s.s. (G1) / E. canadensis (G6) 100% 0% 80% 60% 50% 40% 20% E. granulosus s.s. (G1) E. canadensis (G6)

The multiplex-PCR offers the possibility to avoid DNA One step Multiplex PCR for detection and genotyping of E. granulosus complex II- Multiplex-PCR The multiplex-PCR offers the possibility to avoid DNA extraction steps. Metacestode material: Hydatid fluid, protoscleces (fertile cyst) or cyst layers (germinal- and laminated layer). +++ whole profile of E. granulosus s.s. + and +/- partial profile (only two or one band) - no amplification

Multiplex PCR is a powerful approach for regular One step Multiplex PCR for detection and genotyping of E. granulosus complex III- Conclusion The multiplex-PCR offers three levels of interpretation: genus echinococcus, E. granulosus complex and species. Two targets per species allows: valid identification and avoiding of system failure due to eventual single nucleotide polymorphisms (SNP). The Multiplex PCR showed a high sensitivity (5 ng) and 100 % specificity when tested with other Taenia (T. saginata, T. crassiceps, T. solium, T. pisiformis). Multiplex PCR is a powerful approach for regular worldwide epidemiological control of E. granulosus complex

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One step Multiplex PCR for detection and genotyping of Innovation for the Management of Echinococcosis Besançon, 2014 One step Multiplex PCR for detection and genotyping of E. granulosus complex Ghalia BOUBAKER, Bruno GOTTSTEIN, Markus SPILIOTIS Institute of Parasitology, Vetsuisse Faculty Berne, Langgass-Str. 122, CH-3012 Berne, Switzerland.