Real-time PCR detection and speciation of Cryptosporidium infection using Scorpion probes.
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.
Stroup
Suzanne E
SE
Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA.
Roy
Shantanu
S
Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Dhaka-1000, Bangladesh.
Mchele
John
J
Department of Medicine, Kilimanjaro Christian Medical Centre, PO Box 2222, Moshi, Tanzania.
Maro
Venance
V
Department of Medicine, Kilimanjaro Christian Medical Centre, PO Box 2222, Moshi, Tanzania.
Ntabaguzi
Simon
S
Department of Medicine, Kilimanjaro Christian Medical Centre, PO Box 2222, Moshi, Tanzania.
Siddique
Abdullah
A
Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Dhaka-1000, Bangladesh.
Kang
Gagandeep
G
Department of Gastrointestinal Sciences and Haematology, Christian Medical College and Hospital, Vellore, India.
Guerrant
Richard L
RL
Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA.
Kirkpatrick
Beth D
BD
University of Vermont College of Medicine, Burlington, VT 05401, USA.
Fayer
Ronald
R
US Department of Agriculture, Beltsville, MD 20705, USA.
Herbein
Joel
J
Techlab, Inc., Blacksburg, VA 24060, USA.
Ward
Honourine
H
Department of Medicine, Tufts University, Boston, MA 02111, USA.
Haque
Rashidul
R
Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Dhaka-1000, Bangladesh.
Houpt
Eric R
ER
Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA.
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
D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S