Differentiation of Cryptosporidium parvum, C. muris, and C. baileyi by PCR-RFLP analysis of the 18S rRNA gene.
We have developed a combined polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay for the detection and differentiation of Cryptosporidium parvum, Cryptosporidium muris, and Cryptosporidium baileyi. The assay utilizes PCR to amplify an approximately 1750 basepair product of the Cryptosporidium 18S rRNA gene. Following double digestion with restriction endonucleases Dral and Vsp1, this PCR product yields DNA fragments that can be resolved electrophoretically into RFLP profiles that are distinct for C. parvum, C. muris, and C. baileyi. Previous PCR-restriction analyses could not simultaneously differentiate all three species. Future application of this technique could include predicting the disease-causing potential of oocyst-contaminated environmental specimens and helping to determine the source of oocyst contamination.
Leng
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Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan 66506-5605, USA.
Mosier
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Oberst
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D000818:Animals; D001483:Base Sequence; D003458:Cryptosporidium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D016785:Cryptosporidium parvum / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D015183:Restriction Mapping
D017931:DNA Primers; D012337:RNA, Ribosomal, 18S; C052712:endodeoxyribonuclease VspI; D015252:Deoxyribonucleases, Type II Site-Specific; C035594:TTTAAA -specific type II deoxyribonucleases