title: Real-time PCR detection and speciation of Cryptosporidium infection using Scorpion probes.
abstract: 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 10(3) 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.
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Suzanne E
authors.initials: SE
authors.lastname: Stroup
authors.affiliation: Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Dhaka-1000, Bangladesh.
authors.forename: Shantanu
authors.initials: S
authors.lastname: Roy
authors.affiliation: Department of Medicine, Kilimanjaro Christian Medical Centre, PO Box 2222, Moshi, Tanzania.
authors.forename: John
authors.initials: J
authors.lastname: Mchele
authors.affiliation: Department of Medicine, Kilimanjaro Christian Medical Centre, PO Box 2222, Moshi, Tanzania.
authors.forename: Venance
authors.initials: V
authors.lastname: Maro
authors.affiliation: Department of Medicine, Kilimanjaro Christian Medical Centre, PO Box 2222, Moshi, Tanzania.
authors.forename: Simon
authors.initials: S
authors.lastname: Ntabaguzi
authors.affiliation: Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Dhaka-1000, Bangladesh.
authors.forename: Abdullah
authors.initials: A
authors.lastname: Siddique
authors.affiliation: Department of Gastrointestinal Sciences and Haematology, Christian Medical College and Hospital, Vellore, India.
authors.forename: Gagandeep
authors.initials: G
authors.lastname: Kang
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Guerrant
authors.affiliation: University of Vermont College of Medicine, Burlington, VT 05401, USA.
authors.forename: Beth D
authors.initials: BD
authors.lastname: Kirkpatrick
authors.affiliation: US Department of Agriculture, Beltsville, MD 20705, USA.
authors.forename: Ronald
authors.initials: R
authors.lastname: Fayer
authors.affiliation: Techlab, Inc., Blacksburg, VA 24060, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Herbein
authors.affiliation: Department of Medicine, Tufts University, Boston, MA 02111, USA.
authors.forename: Honourine
authors.initials: H
authors.lastname: Ward
authors.affiliation: Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Dhaka-1000, Bangladesh.
authors.forename: Rashidul
authors.initials: R
authors.lastname: Haque
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
mesh_terms: D000818:Animals; D001483:Base Sequence; D003457:Cryptosporidiosis / Q000469:parasitology*; D003458:Cryptosporidium / Q000145:classification* / Q000302:isolation & purification*; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D005243:Feces / Q000469:parasitology; D020459:Genes, rRNA; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S