Skip to Main Content

The results of qPCR assays using the DNA standard with or without IMBs are shown in Table 1. In the absence of Tween 20, the Ct values of the untreated IMBs and the positive control were 23.09 and 23.14, respectively, indicating that a 10% suspension of IMBs did not inhibit the qPCR assay. DNA isolated from the IMBs after SET without Tween 20 was not amplified (Table 1). In our previous study, the final concentration of SDS in the PCR mix was 0.01%, which inhibits PCR (Sekikawa & Toshiki 2015). Further, we reported that inhibition of PCR by 0.01% SDS is reduced by adding 5% Tween 20 (Sekikawa & Toshiki 2015). Therefore, each PCR assay was performed in the presence of Tween 20 to examine the effect on DNA amplification of the IMBs after they were subjected to SET. Here, we show that Tween 20 abolished the inhibitory effect of the IMBs after SET (Table 1). The results of the Ct values in the presence of 5% Tween 20 indicate that the activity of Taq polymerase was not affected by SET.

Table 1

qPCR assays of DNA extracted from IMBs subjected to SET

  Ct value (mean ± SE)
 Conc. of SDS inConc. of Tween 20 in the PCR mix
Sample for PCRthe PCR mix0%5%
IMBs and standard DNA 0.0% 23.09 ± 0.07 23.42 ± 0.01 
IMBs after SET and standard DNA 0.01% ND 23.26 ± 0.05 
Standard DNA (positive control) 0.0% 23.14 ± 0.02 23.28 ± 0.07 
  Ct value (mean ± SE)
 Conc. of SDS inConc. of Tween 20 in the PCR mix
Sample for PCRthe PCR mix0%5%
IMBs and standard DNA 0.0% 23.09 ± 0.07 23.42 ± 0.01 
IMBs after SET and standard DNA 0.01% ND 23.26 ± 0.05 
Standard DNA (positive control) 0.0% 23.14 ± 0.02 23.28 ± 0.07 

qPCR, quantitative real-time PCR; IMB, immunomagnetic bead; SET, surfactant extraction treatment; SE, standard error; ND, none detected. n = 3. Standard DNA (3 × 104 copies per reaction) was used for qPCR. The DNA standard included a partial sequence of the 18S rRNA gene of C. parvum.

Close Modal

or Create an Account

Close Modal
Close Modal