A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex

Abstract

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

Figure 1. Genotype profile of the E. granulosus complex by mPCR.

(A) Schematic representation of the genotype specific banding patterns amplified by mPCR: (lane 1) E. granulosus s.s. (G1/G2/G3), (lane 2) E. equinus (G4), (lane 3) E. ortleppi (G5), (lane 4) E. canadensis (G6/G7), (lane 5) E. canadensis (G8/G10), (lane 6) E. multilocularis and (lane 7) E. vogeli. The product sizes are specified in bp and the corresponding genes are shown in Table 1. (B) Result of a mPCR using 5 ng of purified template DNA of the known Echinococcus species described above (lanes 1–7) visualized on a 2% agarose gel. The target of 1232 bp is specific for the Echinococcus genus and is also amplified for E. multilocularis (lane 6) and E. vogeli (lane 7). The 110 bp band allows specific detection of E. granulosus complex members (lanes 1–5). All bands between 1232 bp and 110 bp specifically detected one E. granulosus complex species/genotype and showed no cross-reactivity with other members. (C) Specificity test of the mPCR for the genus Echinococcus and other closely related cestodes of the family; E. granulosus (G1/G2/G3) (lane 1), E. multilocularis (lane 2), E. vogeli (lane 3), T. saginata (lane 4), T. solium (lane 5), T. crassiceps (lane 6), T. taeniaformis (lane 7) and T. pisiformis (lane 8). The expected banding pattern was observed for E. granulosus (G1/G2/G3) (lane 1), E. multilocularis (lane 2) and E. vogeli (lane 3) and no PCR products were detected for the Taenia samples. N: PCR-negative control (ddH₂O). M: 100-bp DNA ladder (Promega).

Figure 3. Detection limit of one species in a dual-species DNA mixture.

A) To mimic a mixture of different Echinococcus granulosus complex members, as it can occur in egg-derived samples, DNA from E. granulosus s.s. (G1) and E. canadensis (G6) was mixed in different ratios and the mPCR was performed using 250 ng (lanes 1–5), 50 ng (lanes 6–9) or 5 ng (lanes 10–16) DNA template. The detection limit of one species in a dual-species DNA mixture was measured at 5% (lane 4, 250 ng template DNA), 2.5% (lane 6, 50 ng template DNA) and 20% (lanes 11 and 15, each 5 ng template DNA). B) To test if all 11 targets can be amplified in parallel, 5 ng template DNAs from E. granulosus s.s. (G1), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6) and E. canadensis (G10) were mixed and used together in one single mPCR. All targets were successfully amplified and no missing or non-specific amplicon was detected (lane 1). Lane 2 shows the virtual banding pattern. Amplicon sizes and genotype specificities are marked on the left side. M: 100-bp DNA ladder (Promega).

Figure 2. Specificity of the mPCR approach based on number of cycles.

(A–C) Different quantities of E. granulosus s.s. (G1) DNA were used as templates in the mPCR: 5 ng (lane 1), 25 ng (lane 2), 50 ng (lane 3), 100 ng (lane 4) and 250 ng (lane 5). The mPCR was run with 25 cycles (A), 30 cycles (B) or 35 cycles (C) of amplification. For the E. granulosus s.s. (G1) template, the genotype was clearly detectable in all setups, but performing the mPCR with 30 or 35 cycles resulted in a visible background smear and some very light additional bands. A reduced setup was performed for the other genotypes (D). The mPCR was run with 5 ng (lanes 1, 3, 5, 7 and 9) or with 250 ng (lanes 2, 4, 6, 8 and 10) and 30 cycles of amplification. In contrast to E. granulosus s.s. (G1/G2/G3) (lanes 1 and 2) which showed only minor unspecific products, the mPCR amplified unspecific products for E. equinus (G4) (lanes 3 and 4), E. ortleppi (G5) (lanes 5 and 6), E. canadensis (G6/G7) (lanes 7 and 8) and E. canadensis (G8/G10) (lanes 9 and 10). Thus, additional numbers of PCR cycles result in unspecific PCR products hampering the readout. M: 100-bp DNA ladder (Promega).

Figure 4. Specificity of the mPCR approach in contaminated samples.

To mimic host derived contaminations of the template DNA, 5 ng of E. granulosus s.s. (G1) DNA was mixed in different ratios (1∶1 lane 1, 1∶2 lane 2, 1∶5 lane 3, 1∶10 lane 4, 1∶20 lane 5, 1∶30 lane 6, 1∶ 40 lane 7, 1∶ 50 lane 8, 1∶100 lane 9 and 1∶ 200 lane 10) with (A) DNA extracted from feces of a helminth-infection free dog and (B) calf thymus DNA. The background smear increased by applying more foreign DNA, but the genotype was still detectable, even when 1 µg total DNA was used as template (lane 9). M: 100-bp DNA ladder (Promega).

Figure 5. Direct mPCR on frozen and fixed E. granulosus complex material.

(A) 1 µl (lane 1) or 2 µl (lane 2) of previously frozen hydatid fluid aspirated from an equid cyst was used directly in the mPCR without resulting in genotype specific PCR products. In parallel, 1 ml of the hydatid fluid was boiled for 30 min followed by a centrifugation step. Different volumes of the resulting supernatant were used in the mPCR (0.25 µl lane 3, 0.5 µl lane 4, 1 µl lane 5, 1.5 µl lane 6, 2 µl lane 7, 2.5 µl lane 8, 3 µl lane 9, 10 µl lane 10). Note that using 1–3 µl resulted in the detection of E. equinus (G4), although with some minor additional background amplicons. Frozen (B) and EtOH-fixed (C) E. granulosus s.s. (G1) protoscoleces were treated by alkaline lysis and the supernatant was used without (lanes 1 and 2) or with dilution (1∶1 lane 3, 1∶2 lane 4, 1∶4 lane 5, 1∶6 lane 6, 1∶8 lane 7, 1∶10 lane 8, 1∶25 lane 9) for mPCR. Undiluted supernatant (1 µl lane 1 and 2 µl lane 2) resulted in failed mPCR in both setups. If 2 µl of diluted supernatant was used for mPCR, genotyping was successfully performed for frozen protoscoleces on dilution ratios of 1∶8 to 1∶10 and for EtOH-fixed protoscoleces on ratios between 1∶2 to 1∶4. M: 100-bp DNA ladder (Promega).