Abstract
Cryptosporidium is a protozoan that causes gastrointestinal disease. Extraction of DNA from Cryptosporidium oocysts is complicated due to the hard outer wall. Generally, the oocyst DNA is extracted with proteinase K in a lysis buffer containing sodium dodecyl sulfate (SDS). However, SDS, being a strong inhibitor of Taq and Bst DNA polymerases, needs to be removed before PCR and Loop-mediated isothermal amplification (LAMP), respectively. In this study, we evaluated the ability of nonionic surfactants to suppress the inhibition of Bst DNA polymerase by anionic surfactants such as SDS, in the LAMP method. The primer set for LAMP was designed from the polythreonine gene of C. parvum. Bst DNA polymerase used in LAMP was inhibited by 0.01% of SDS or sodium dodecyl benzene sulfonate (SDBS), but this inhibition was suppressed by the addition of 5% of Triton X－100 or Tween 20. Subsequently, we applied this suppression of Bst DNA polymerase inhibition to C. parvum DNA detection using the LAMP method and succeeded in detecting DNA from ten oocysts in the presence of 0.01% SDS or SDBS.
