Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water.
Eight pairs of published PCR primers were evaluated for the specific detection of Cryptosporidium parvum and Giardia lamblia in water. Detection sensitivities ranged from 1 to 10 oocysts or cysts for purified preparations and 5 to 50 oocysts or cysts for seeded environmental water samples. Maximum sensitivity was achieved with two successive rounds of amplification and hybridization, with oligonucleotide probes detected by chemiluminescence. Primer annealing temperatures and MgCl2 concentrations were optimized, and the specificities of the primer pairs were determined with closely related species. Some of the primers were species specific, while others were only genus specific. Multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was demonstrated with primers amplifying 256- and 163-bp products from the 18S rRNA gene of Cryptosporidium and the heat shock protein gene of Giardia, respectively. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.
Rochelle
P A
PA
Water Quality Laboratory, Metropolitan Water District of Southern California, La Verne 91750-3399, USA. prochelle@mwd.dst.ca.us
De Leon
R
R
Stewart
M H
MH
Wolfe
R L
RL
D000818:Animals; D001483:Base Sequence; D016785:Cryptosporidium parvum / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D017125:Genes, Protozoan; D016829:Giardia lamblia / Q000235:genetics* / Q000302:isolation & purification*; D006360:Heat-Shock Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D015800:Protozoan Proteins / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature; D014867:Water / Q000469:parasitology*
D017931:DNA Primers; D006360:Heat-Shock Proteins; D015800:Protozoan Proteins; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S; D014867:Water