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J Med Microbiol 55 (2006), 1217-1222; DOI: 10.1099/jmm.0.46678-0
© 2006 Society for General Microbiology
ISSN 1473-5644

Real-time PCR detection and speciation of Cryptosporidium infection using Scorpion probes

Suzanne E. Stroup1, Shantanu Roy2, John Mchele3, Venance Maro3, Simon Ntabaguzi3, Abdullah Siddique2, Gagandeep Kang4, Richard L. Guerrant1, Beth D. Kirkpatrick5, Ronald Fayer6, Joel Herbein7, Honourine Ward8, Rashidul Haque2 and Eric R. Houpt1

1 Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA

2 Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Dhaka-1000, Bangladesh

3 Department of Medicine, Kilimanjaro Christian Medical Centre, PO Box 2222, Moshi, Tanzania

4 Department of Gastrointestinal Sciences and Haematology, Christian Medical College and Hospital, Vellore, India

5 University of Vermont College of Medicine, Burlington, VT 05401, USA

6 US Department of Agriculture, Beltsville, MD 20705, USA

7 Techlab, Inc., Blacksburg, VA 24060, USA

8 Department of Medicine, Tufts University, Boston, MA 02111, USA

Correspondence
Eric R. Houpt
erh6k{at}virginia.edu

Received 12 April 2006
Accepted 6 June 2006

At least eight species of Cryptosporidium can cause human infection and disease. A real-time PCR (qPCR) assay based on the 18S rRNA gene and utilizing a Scorpion probe was developed to detect all human-pathogenic Cryptosporidium without the usual need for nested amplification. Sensitivity of detection in stool samples was highest using a glass bead-based DNA extraction method (under 103 oocysts per stool sample). The assay was validated against 123 human stool specimens from Bangladesh and Tanzania, exhibited a sensitivity and specificity of >91 % versus microscopy, and detected an additional eight microscopy-negative infections. Cryptosporidium parvum-specific and Cryptosporidium meleagridis-specific Scorpion qPCR assays that provided 100 % accurate speciation compared with VspI RFLP analysis and sequencing were developed subsequently. These Scorpion probe qPCR assays are simpler to perform than existing nested PCR and RFLP methods for diagnosis and epidemiological investigation of cryptosporidiosis.




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