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Oocysts were recovered from samples of distilled water or river water using IMS, and DNA was extracted from IMB–oocyst conjugates using SET. The limit of detection of the qPCR assay was two oocysts/PCR reaction mixture (Table 3). The Ct values without Tween 20 at the dilution ratio of 1 were not detected because the concentrations of SDS in the PCR mix were 0.01%. The PCR assay is not inhibited by 0.001% SDS (Sekikawa & Toshiki 2015). Therefore, the amplification of DNA in 10 L distilled water with Tween 20 using a 10−1 dilution was not detected because the concentration of SDS was 0.001%.

Table 3

qPCR detection of oocyst DNA in a 10 L water sample spiked with oocysts that were collected using IMS–SET

  Ct value (mean ± SE)
Theoretical number ofDistilled water (DW) sample Conc. of Tween 20 in the PCR mix
River water sample Conc. of Tween 20 in the PCR mix
Dilution of sample for PCRoocysts in the PCR mix0%5%0%5%
2000 NDa 25.64 ± 0.17 NDa 26.04 ± 0.20 
10−1 200 29.28 ± 0.17 29.01 ± 0.15 ND 29.39 ± 0.20 
10−2 20 32.46 ± 0.14 32.30 ± 0.16 32.80 ± 0.17 32.70 ± 0.18 
10−3 36.32 ± 0.22 35.79 ± 0.25 36.31 ± 0.27 36.19 ± 0.23 
10−4 0.2 ND ND ND ND 
  Ct value (mean ± SE)
Theoretical number ofDistilled water (DW) sample Conc. of Tween 20 in the PCR mix
River water sample Conc. of Tween 20 in the PCR mix
Dilution of sample for PCRoocysts in the PCR mix0%5%0%5%
2000 NDa 25.64 ± 0.17 NDa 26.04 ± 0.20 
10−1 200 29.28 ± 0.17 29.01 ± 0.15 ND 29.39 ± 0.20 
10−2 20 32.46 ± 0.14 32.30 ± 0.16 32.80 ± 0.17 32.70 ± 0.18 
10−3 36.32 ± 0.22 35.79 ± 0.25 36.31 ± 0.27 36.19 ± 0.23 
10−4 0.2 ND ND ND ND 

qPCR, quantitative real-time PCR; SE, standard error; IMS, immunomagnetic separation; SET, surfactant extraction treatment; ND, none detected.

a0.01% SDS. n = 3. IMS was used to recover oocysts from 10 L of DW or river water spiked with 105 oocysts.

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