A new method for efficient detection of Cryptosporidium RNA by real-time reverse transcription-PCR with surfactants

Cryptosporidium is one of the most common causes of waterborne diseases worldwide. Its oocysts possess a robust wall that is extremely resistant to the chlorine used for potable water disinfection. The current procedures of nucleic acid extraction and purification, such as the freeze–thaw (F/T) method and the commercial kits, are time consuming and expensive. To this end, a surfactant extraction treatment (SET) was developed as a method to extract nucleic acids from Cryptosporidium using only surfactants. The use of 18S rRNA improves the sensitivity of Cryptosporidium detection for real-time polymerase chain reaction (PCR), because 18S rRNA molecules are constitutively present in high copy numbers. Therefore, we applied SET to the detection of Cryptosporidium 18S rRNA using reverse transcription (RT)-PCR for the first time. RT-PCR was inhibited by 0.01% of the anionic surfactant sodium dodecyl sulfate (SDS), whereas the inhibition did not occur with 5% of the nonionic surfactants Tween 20, Triton X-100, Tween 80, and Triton X-114. However, the nonionic surfactants could not completely suppress the inhibition induced by 0.1% SDS. We successfully extracted 18S rRNA genes from oocysts by SET without the F/T method and detected them by real-time RT-PCR. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/ws.2015.063 Takahiro Sekikawa (corresponding author) Kosuke Toshiki Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52–1 Yada, Suruga-ku, Shizuoka 422-8526, Japan E-mail: [email protected]