1. 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.

3. 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)

4. 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).

5. 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

6. 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

7. 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

8. 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)

9. 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

10. 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

11. Thank you for your attention

12. 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.