GIARDIA-INTESTINALIS - DETECTION OF MAJOR GENOTYPES BY RESTRICTION ANALYSIS OF GENE AMPLIFICATION PRODUCTS

The polymerase chain reaction (PCR) has been used to amplify a 0. 52 k b segment of Giardia intestinalis DNA, using primers specific for nucl eotide sequences conserved within two genes (tsp11 and tsa417) that en code homologous, cysteine-rich trophozoite surface proteins. Using pro ducts amplified from axenic isolates belonging to genetic groups I and II (defined on the basis of allozyme electrophoresis data), restricti on endonuclease analysis revealed both tsp11-like and tsa417-like frag ments within all samples. The study also identified among the amplific ation products of group II organisms an additional fragment, containin g a novel PstI site, that is not detected in the reaction products of group I isolates. The recovery of three distinct PCR products from eac h group II isolate was verified by cloning the fragments into the plas mid vector pGEM-7. Fragments containing the new PstI site possess the ClaI site common to both tsp11 and tsa417-like fragments, but they lac k the HindIII site which characterizes tsp11-like fragments and also l ack the PstI and KpnI sites which characterize tsa417-like fragments. Spot-blot analyses using cloned fragments of all three types as probes showed strong homologous hybridization but weak heterologous hybridiz ation, indicating that each type differs substantially in nucleotide s equence from the others. Because the samples of Giardia DNA used in th e PCR were purified from cultures that had been established from singl e trophozoites, the data indicate that individual trophozoites belongi ng to genetic group II possess three homologous genes defined by these related fragments. The presence of a PstI site in the amplified segme nt of the newly-discovered third gene of group II organisms provides a simple diagnostic means of differentiating group I and II isolates.