DETECTION OF GIARDIA IN ENVIRONMENTAL WATERS BY IMMUNO-PCR AMPLIFICATION METHODS

Genomic DNA was extracted either directly from Giardia muris cysts see ded into environmental surface waters or from cysts isolated by immuno magnetic beads (IMB). A 0.171-kbp segment of the giardin gene was PCR- amplified following ''direct extraction'' of Giardia DNA from seeded C ahaba river water concentrate with moderate turbidity (780 JTU's), but DNA purified from seeded Colorado river water concentrates with high turbidity (2 X 10(5) JTUs) failed to amplify. However, if the cysts we re first separated by the IMB approach from seeded Cahaba or Colorado river waters, and the DNA released by a freeze-boil Chelex(R)100 treat ment, detection of G. muris by PCR amplification could be achieved at a sensitivity of 3 x 10(0) or 3 x 10(1) cysts/ml, respectively. If, ho wever, the G. muris cysts used to seed even moderately turbid river wa ters (780 JTUs) were formalin treated (which is conventionally used fo r microscopic examination), neither direct extraction nor IMB purifica tion methods yielded amplifiable DNA. Use of immunomagnetic beads to s eparate Giardia cysts from complex matrices of environmental surface w aters followed by DNA release and PCR amplification of the target giar din gene improved the reliability of detection of this pathogen with t he required sensitivity.