AWWA QTC98335

This paper describes a rapid and simple polymerase chain reaction (PCR) protocol for the detection of Cryptosporidium parvum. This protocol uses PCR reagents which have been prepared in tablet form and PCR primers specific for Cryptosporidium parvum. The use of PCR reagent tablets reduces time and labor required to prepare PCR reagent cocktails, increases reproducibility, and decreases the potential for contamination. Each PCR assay also contains a thermostable DNA intercalating fluorescent dye. Endpoint detection of PCR product is performed using a fluorometer which accommodates PCR tubes, providing a closed tube assay which does not require gel electrophoresis analysis of PCR products. Since the tubes containing the completed reactions do not need to be opened the chance of laboratory contamination with PCR product is greatly reduced. Endpoint detection using the fluorometer currently has a detection limit of five oocysts using a purified oocyst stock and template DNA preparation by freeze-thaw cycling. A detection limit of ten oocysts was obtained for seeded PCR assays containing immunomagnetic separation (IMS) purified environmental raw water concentrates (equivalent to 0.5 mL packed pellet per PCR). Future development of the kit may incorporate fluorescent probe-based technology for the detection of specific PCR product which may allow the detection of a single C. parvum oocyst.

Product Details

Published:

01/01/1998

ISBN(s):

0898679796

Number of Pages:

7

File Size:

1 file , 100 KB