非イオン界面活性剤のPCR阻害抑制効果を用いたクリプトスポリジウムDNAの検出

抄録

Cryptosporidium is a protozoan that causes gastrointestinal disease and is resistant to chlorine disinfection. The method of extracting Cryptosporidium DNA from the hard outer shell, called an oocyst, of Cryptosporidium at a high yield is complicated. Generally, the DNA is extracted using proteinase K in a lysis buffer containing sodium dodecyl sulfate (SDS). However, SDS needs to be removed for PCR, because it is one of the strong inhibitors of PCR. We investigated a simple method for suppressing PCR inhibition with nonionic surfactants for the detection of C. parvum DNA. The primer set for real-time PCR was designed from the polythreonine gene of C. parvum. PCR was inhibited by 0.01% of SDS or Sodium dodecyl benzene sulfonate (SDBS), but the inhibition was reversed with the addition of 0.5% Tween 20. Subsequently, we applied this suppression method to C. parvum DNA detection, and succeeded in detecting DNA from 10 oocysts by PCR in the presence of 0.01% SDS or SDBS.