Differentiation of major genotypes ofGiardia intestinalisby polymerase chain reaction analysis of a gene encoding a trophozoite surface antigen

Abstract

The polymerase chain reaction (PCR) has been used to amplify in vitro a semi-conserved region of a gene encoding an M(r) 68-72,000 surface antigen of Giardia intestinalis trophozoites. Using primers specific for conserved nucleotide sequences identified within the promoter-distal portion of two homologous genes (tsp11 and tsa417) cloned previously from the G. intestinalis isolates Ad-1 (from Australia) and WB (from Afghanistan), a single PCR-amplified DNA fragment of the expected size (0.52 kilobases) was obtained in high yield from either purified DNA or whole trophozoites of the Ad-1 isolate and from every 1 to 9 other axenic G. intestinalis isolates belonging to genetic groups I and II (defined previously on the basis of allozyme electrophoresis data--Andrews et al. 1989). Discernible product was recovered from as few as 2-4 trophozoites. In contrast, 6 G. intestinalis isolates that were assigned by allozymic analysis to genetic groups III/IV yielded small amounts of a 0.37-kilobase (kb) amplification product (with evidence in some samples of an additional 0.4 or 0.18 kb fragment) but no 0.52 kb product. Two animal-derived isolates of G. duodenalis (one from an Australian native rodent, Notomys alexis, the other from a domestic cat) also yielded a single 0.37 kb PCR-amplified fragment, whereas an isolate from another cat produced a 0.34 kb fragment. No product was recovered from G. muris, a morphologically distinct species of Giardia. The results demonstrate that different genotypes of G. duodenalis can be distinguished using this assay and that it is diagnostic for isolates belonging to two major clusters (groups I/II and III/IV) of G. intestinalis. The amplified DNA segment appears to be relatively conserved among group I and group II isolates of G. intestinalis. A related but clearly distinct sequence seems to be conserved among group III/IV isolates of G. intestinalis and some isolates of G. duodenalis.