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The Ct values obtained using oocysts and the conjugates are shown in Table 2. DNA was not detected following extraction from the oocysts or from the conjugates subjected to SET. SDS inhibited the PCR assay because the concentration of SDS in the PCR tube was 0.01%. However, it was difficult to judge whether the SET extracted DNA from the conjugates. To test whether Tween 20 suppressed the inhibition of the PCR assay by SDS, we determined the effect of combining the conjugates after SET and Tween 20. It was found that in the presence of Tween 20, the DNA extracted from the conjugates using SET was amplified and that the mean Ct value of the conjugates (25.32) was not significantly different compared with that of the oocysts recovered using centrifugation (25.27) (Table 2). These results indicate that DNA can be extracted from conjugates and amplified after SET.

Table 2

Effect of IMB–oocyst conjugates combined with SET on the qPCR assay

  Ct value (mean ± SE)
Sample for DNADNA extractionConc. of Tween 20 in the PCR mix
extractionmethod0%5%
IMB–oocyst conjugates SET NDa 25.32 ± 0.03 
Oocysts SET NDa 25.27 ± 0.06 
Oocysts F/T, DNA purification kit 25.64 ± 0.02 25.70 ± 0.03 
  Ct value (mean ± SE)
Sample for DNADNA extractionConc. of Tween 20 in the PCR mix
extractionmethod0%5%
IMB–oocyst conjugates SET NDa 25.32 ± 0.03 
Oocysts SET NDa 25.27 ± 0.06 
Oocysts F/T, DNA purification kit 25.64 ± 0.02 25.70 ± 0.03 

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

a0.01% SDS. n = 4. Oocysts were recovered from 10 mL of distilled water (DW) spiked with 105 oocysts using immunomagnetic separation (IMS) or centrifugation. Oocyst DNA was extracted using SET or F/T combined with a DNA purification kit.

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