Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts.
A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.
Mahbubani
M H
MH
Department of Biology, University of Louisville, Kentucky 40292.
Bej
A K
AK
Perlin
M
M
Schaefer
F W
FW
Jakubowski
W
W
Atlas
R M
RM
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003598:Cytoskeletal Proteins / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005872:Giardia / Q000302:isolation & purification* / Q000378:metabolism / Q000472:pathogenicity; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics; D012680:Sensitivity and Specificity
D003598:Cytoskeletal Proteins; D016054:DNA, Protozoan; D015800:Protozoan Proteins; C038207:giardin protein, Giardia lamblia