Cryptosporidium Diagnostic Assays: Molecular Detection

  • Guy Robinson
  • Kristin Elwin
  • Rachel M. ChalmersEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2052)


Molecular diagnostic assays for Cryptosporidium are usually based on PCR and may detect the entire genus or target specified species. Of the ~40 species, fewer than half have been reported from humans, and most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or Cryptosporidium hominis. Here we describe a nested PCR for the detection of all Cryptosporidium spp. that can then be differentiated by sequencing the PCR amplicons, and a duplex, real-time PCR for the simultaneous detection and differentiation of C. parvum and C. hominis.


Cryptosporidium C. parvum C. hominis Detection DNA extraction Nested PCR Real-time PCR 



The research leading to the development of the duplex real-time PCR described here has received funding from the European Union Seventh Framework Program [(FP7/2007-2013) (FP7/2007-2011)] under Grant agreement no: 311846.


  1. 1.
    Smith H (2008) Diagnostics. In: Fayer R, Xiao L (eds) Cryptosporidium and cryptosporidiosis, 2nd edn. CRC Press, Boca RatonGoogle Scholar
  2. 2.
    Chalmers RM, Campbell BM, Crouch N, Charlett A, Davies AP (2011) Comparison of the diagnostic sensitivity and specificity of seven Cryptosporidium assays used in the United Kingdom. J Med Microbiol 60:1598–1604CrossRefGoogle Scholar
  3. 3.
    Wiedenmann A, Kruger P, Botzenhart K (1998) PCR detection of Cryptosporidium parvum in environmental samples - a review of published protocols and current developments. J Ind Microbiol Biotechnol 21:150–166CrossRefGoogle Scholar
  4. 4.
    Binnicker MJ (2015) Multiplex molecular panels for diagnosis of gastrointestinal infection: performance, result interpretation, and cost-effectiveness. J Clin Microbiol 53:3723–3728CrossRefGoogle Scholar
  5. 5.
    Jiang J, Alderisio KA, Xiao L (2005) Distribution of Cryptosporidium genotypes in storm event water samples from three watersheds in New York. Appl Environ Microbiol 71:4446–4454CrossRefGoogle Scholar
  6. 6.
    Xiao L, Escalante L, Yang C, Sulaiman I, Escalante AA, Montali RJ, Fayer R, Lal AA (1999) Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl Environ Microbiol 65:1578–1583CrossRefGoogle Scholar
  7. 7.
    Xiao L, Alderisio K, Limor J, Royer M, Lal AA (2000) Identification of species and sources of Cryptosporidium oocysts in storm waters with a small-subunit rRNA-based diagnostic and genotyping tool. Appl Environ Microbiol 66:5492–5498CrossRefGoogle Scholar
  8. 8.
    Hadfield SJ, Robinson G, Elwin K, Chalmers RM (2011) Detection and differentiation of Cryptosporidium spp. in human clinical samples by use of real-time PCR. J Clin Microbiol 49:918–924CrossRefGoogle Scholar
  9. 9.
    Tosini F, Drumo R, Elwin K (2010) The CpA135 gene as a marker to identify Cryptosporidium species infecting humans. Parasitol Int 59:606–609CrossRefGoogle Scholar
  10. 10.
    Moore CE, Elwin K, Phot N, Seng C, Mao S, Suy K, Kumar V, Nader J, Bousfield R, Perera S, Bailey JW, Beeching NJ, Day NP, Parry CM, Chalmers RM (2016) Molecular characterisation of Cryptosporidium species and Giardia duodenalis from symptomatic Cambodian children. PLoS Negl Trop Dis 10:e0004822CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Guy Robinson
    • 1
  • Kristin Elwin
    • 1
  • Rachel M. Chalmers
    • 1
    Email author
  1. 1.Cryptosporidium Reference Unit, Public Health Wales Microbiology and Health ProtectionSingleton HospitalSwanseaUK

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