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Title: SPECIES SPECIFIC REAL-TIME PCR FOR CRYPTOSPORIDIUM IN STOOL USING SCORPION PROBES

Author
item STROUP, SUZANNE - UNIVERSITY OF VIRGINIA
item SHANTANU, ROY - DHAKA-1000, INDIA
item MCHELE, JOHN - CHRISTIAN M.C.,TANZANIA
item MARO, VENANCE - CHRISTIAN M.C.,TANZANIA
item NTABAGUZI, SIMON - CHRISTIAN M.C., TANZANIA
item SIDDIQUE, ABDULLAH - DHAKA-1000, INDIA
item KANG, GAGANDEEP - C.M.C. VELLORE, INDIA
item GUERRANT, RICHARD - UNIVERSITY OF VIRGINIA
item KIRPATRICK, BETH - UNIVERSITY OF VERMONT
item Fayer, Ronald
item HERBEIN, JOEL - TECHLAB, INC., VIRGINIA
item WARD, HONORINE - TUFTS UNIVERSITY, MA
item HAQUE, RASHIDUL - DHAKA-1000, INDIA
item HOUPT, ERIC - UNIVERSITY OF VIRGINIA

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/6/2006
Publication Date: 7/10/2006
Citation: Stroup, S.E., Shantanu, R., Mchele, J., Maro, V., Ntabaguzi, S., Siddique, A., Kang, G., Guerrant, R.L., Kirpatrick, B.D., Fayer, R., Herbein, J., Ward, H., Haque, R., Houpt, E.R. 2006. Species specific real-time PCR for Cryptosporidium in stool using scorpion probes. Journal of Clinical Microbiology. E-publication:Volume 55.

Interpretive Summary: Cryptosporidiosis is a diarrheal disease of humans and animals caused by protozoan parasites in the genus Cryptosporidium. There are now 17 named species and many unnamed genotypes some of which infect humans and others that have been found only in specific animals. Molecular tools are required to identify each of these species/genotypes in order trace back to the source and thereby determine the cause or routes of transmission. The present manuscript compares methods for recovering DNA from several species and using newly developed probes to quickly identify those most frequently found in human infections.

Technical Abstract: Real-time PCR assays based on the 18S rRNA gene were developed utilizing Scorpion probes to speciate Cryptosporidium oocysts in fecal specimens. The first Scorpion PCR detected oocysts at a sensitivity of 1000 oocysts per stool sample without the need for nested amplification at a sensitivity and specificity of 92 and 91% versus microscopy and 83 and 95% versus antigen detection on 142 human stools. A second method was developed to identify Cryptosporidium parvum and C. meleagridis specific Scorpion PCR reactions that were 100% accurate versus Vsp 1 RFLP. This method revealed the three most frequently found species causing human cryptosporidiosis individually and mixed in stools, indicating its usefulness for diagnosis and epidemiological investigations.