title: A simple method for extracting DNA from Cryptosporidium oocysts using the anionic surfactant LSS.
abstract: Detection of low amounts of Cryptosporidium oocysts in raw water sources is considered an important component in the management, prevention and control of Cryptosporidium in drinking water supplies as Cryptosporidium causes massive waterborne outbreaks worldwide. As Cryptosporidium has a robust oocyst that is extremely resistant to chlorine and other drinking water disinfectants, both the freeze-thaw method and DNA extraction kits have been commonly used for extracting and purifying DNA from the oocyst. However, the DNA extraction procedures are time consuming and costly. Therefore, a simple and low-cost method to extract and purify DNA from the robust oocyst has been required. In this study, we discussed a simple method for detecting Cryptosporidium DNA with the anionic surfactant, n-lauroylsarcosine sodium salt (LSS) using the loop-mediated isothermal amplification (LAMP) to eliminate the need for the freeze-thaw method and the DNA extraction kits. As a result, Bst DNA polymerase was inhibited by 0.1% LSS but not 0.01% LSS and 5% Triton X-100 or Tween 20. Although DNA was extracted from the oocysts by incubating with 0.1% LSS at 90°C for 15 min, Bst DNA polymerase was inhibited by 0.1% LSS. The inhibition by 0.1% LSS was suppressed by adding 5% of the nonionic surfactants, Triton X-100 or Tween 20. The concentration of LSS in a LAMP tube was 0.01% while that in an incubation tube was 0.1%, because LSS in an incubation tube was diluted by a factor of 10 at the DNA amplification process. Therefore, we found that ten oocysts of Cryptosporidium parvum could be detected by incubation with 0.1% LSS, without removing LSS or adding the nonionic surfactants in the LAMP method.
authors.affiliation: Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Japan. sekikawa.shizuokakendai@gmail.com
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sekikawa
authors.forename: Yu
authors.initials: Y
authors.lastname: Kawasaki
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Katayama
authors.forename: Keisuke
authors.initials: K
authors.lastname: Iwahori
mesh_terms: D000818:Animals; D003457:Cryptosporidiosis; D003458:Cryptosporidium / Q000166:cytology* / Q000235:genetics*; D016054:DNA, Protozoan / Q000302:isolation & purification*; D003902:Detergents / Q000737:chemistry*; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors; D033661:Oocysts / Q000737:chemistry* / Q000502:physiology*; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D016054:DNA, Protozoan; D003902:Detergents; D019384:Nucleic Acid Synthesis Inhibitors; C025231:sarkosyl; D012521:Sarcosine