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

Abstract

SUMMARY: The polymerase chain reaction (PCR) has been used to amplifyin vitroa semi-conserved region of a gene encoding anM_r68–72000 surface antigen ofGiardia intestinalistrophozoites. Using primers specific for conserved nucleotide sequences identified within the promoter-distal portion of two homologous genes (tsp11 andtsa417) cloned previously from theG. intestinalisisolates 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 of 9 other axenicG. intestinalisisolates belonging to genetic groups I and II (defined previously on the basis of allozyme electrophoresis data—Andrewset al.1989). Discernible product was recovered from as few as 2–4 trophozoites. In contrast, 6G. intestinalisisolates 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 ofG. 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 fromG. muris, a morphologically distinct species ofGiardia. The results demonstrate that different genotypes ofG. duodenaliscan be distinguished using this assay and that it is diagnostic for isolates belonging to two major clusters (groups I/II and III/IV) ofG. intestinalis. The amplified DNA segment appears to be relatively conserved among group I and group II isolates ofG. intestinalis. A related but clearly distinct sequence seems to be conserved among group III/IV isolates ofG. intestinalisand some isolates ofG. duodenalis.