Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns.
We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research.
Deng
M Q
MQ
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California at Davis, 95616-8743, USA.
Lam
K M
KM
Cliver
D O
DO
D000818:Animals; D001431:Bacteriological Techniques; D001628:Beverages; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology; D002460:Cell Line; D003457:Cryptosporidiosis / Q000469:parasitology / Q000662:veterinary*; D016785:Cryptosporidium parvum / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification* / Q000472:pathogenicity; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005243:Feces / Q000382:microbiology; D019084:Fluorescent Antibody Technique, Indirect; D018189:Immunomagnetic Separation / Q000379:methods*; D008863:Microspheres; D008892:Milk / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D019105:Random Amplified Polymorphic DNA Technique; D019685:Rosales / Q000382:microbiology
D004269:DNA, Bacterial