Detection of Giardia cysts by cDNA probe and application to water samples

Abstract

Giardia is the most common human parasite infection in the United States causing a lengthy diarrhea. Transmission of Giardia is by the fecal-oral route and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia in drinking water through the "Surface Water Treatment Rule." Current methods for detection of Giardia in water rely primarily on microscopic observation of water concentrates by immunofluorescent techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 base pairs long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of M13 vector with insert was isolated from lysed host E. coli XL1- Blue and used for production of the cDNA probe by nick translation with ³²P-labeled nucleotides. Seven different protocols were tested for extracting nucleic acids from the cysts. Using the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates by dot-blot hybridization assays. Environmental concentrates from secondary and tertiary treated sewage or surface waters were screened for Giardia cysts by immunofluorescent and the genespecific probe. Positive signals were observed in sewage and surface water samples without floatation at ten fold greater dilutions than after floatation. It appeared that gene probe detection was slightly more sensitive than microscopic detection of Giardia cysts for wastewater samples. In six surface water samples and two sewage sample no positive results were found either by the cDNA probe or immunofluorescent. Usually, DNA probes are radiolabeled and the most commonly used is ³²P. ³²P is expensive, hazardous and has an extremely short half-life of 14.3 days, necessitating frequent preparation of the nucleic acid probes. Three non-radioactive labeling methods, chemiluminescence, enzyme-linked immunoassay and enhanced chemiluminescence were evaluated. The cDNA probe was labeled by nick translation for chemiluminescence method. Biotinylated deoxyuridine triphosphate was used in place of deoxythymidine triphosphate to produce biotinylated DNA strands. The result of hybridization was visualized by chemiluminescenct detection of DNA. The sensitivity of the chemiluminescent method and the 32P labeled probe was 0.1 pg of DNA in a slot-blot hybridization assay.