Table 1 Detection of Acanthamoeba by real-time PCR (results are indicated as + (positive) or – (negative) for each series of testing)
Treatment of the specimen before DNA amplificationNo. of cysts per ml of suspension (before DNA extraction)
1000 (I)100 (II)30 (III)10 (IV)3 (V)
PCR results (three different series of experiments)
None++++ − −− − −− − −− − −
95°C for 10 min*++++ − −− − −− − −− − −
NaOH 0.1 M†++++ − −− − −− − −− − −
ProtK for 10 min‡++++ − −+ − −− − −− − −
ProtK for 60 min‡++++ − −+ − −− − −− − −
ProtK for 240 min‡+++−+−++−− − −− − −
QIAmp® manual§++++++++−¶−¶ −¶ −¶−¶ −¶ −¶
ProtK for 10 min+QIAmp‡++++++++++ −¶ −¶−¶ −¶ −¶
ProtK for 60 min+QIAmp‡++++++++++ −¶ −¶−¶ −¶ −¶
ProtK for 240 min+QIAmp‡++++++++++ −¶ −¶−¶ −¶ −¶
NaOH 0.1 M†+ QIAmp++++++++−+ − −− − −
MagNA Pure ®**++++++++++ −¶ −¶−¶ −¶ −¶
ProtK for 10 min+MagNA Pure‡+++++++++++++ −¶ −¶
ProtK for 60 min+MagNA Pure‡+++++++++++++ −¶ −¶
ProtK for 240 min+MagNA Pure‡++++++++++ −¶ +−¶ −¶ −¶
NaOH 0.1 M†+MagNA Pure+++++++ − −−+−− − −

  • (I) to (V): concentrations corresponding to 50, 5, 1.5, 0.5 and 0.15 cysts/PCR reaction tube.

  • *Specimens in microtubes were placed in a dry heater for 10 min at 95°C and kept at 20°C for 30 min before amplification; †samples were treated with NaOH (0.1 M final concentration) at 95°C for 5 min; ‡ProtK at 56°C and heat inactivation at 95°C; §QIAmp® DNA Mini Kit (Qiagen)—manual method; ¶true negative values (no delays were observed for the phHV Cts while comparing each result to those obtained with the controls for phHV, indicating the absence of inhibitors; samples showing delays or more than two Cts in comparison with controls were considered inhibited); **MagNA Pure® (Roche)—automatic extraction robot.