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. 2013 Jan 17;7(1):e2017. doi: 10.1371/journal.pntd.0002017

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

Figure 5

(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).