Full Description

In this study, a microwave technique for microsporidia spore lysis and DNA release of E. intestinalis was compared with freeze-thaw techniques prior to DNA extraction. Microwave energy proved to be a simple and efficient method for releasing DNA from microsporidia spores. In addition, previously characterized primers for E. intestinalis, V1 and SI500 (15), were used to design a high fidelity PCR that incorporates dUTP and uracil-N-glycosylase as an extra measure of contamination control. PCR with carryover prevention (PCR-COP) is resistant to false positives resulting from contamination by DNA amplified in previous reactions. Microwave lysis, combined with PCR-COP, produced a 382 bp PCR product with nucleotide sequences that were 100% homologous to published E. intestinalis sequences. The optimized protocol was used to detect seeded E. intestinalis spores in laboratory and in source water. This study was successful in providing a sensitive, specific, high fidelity PCR protocol with carryover prevention for use in human and environmental surveillance of microsporidia. Includes 15 references.

Product Details

Edition: Vol. - No. Published: 01/01/1999File Size: 1 file , 210 KB