PCR-based DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer

The potential for the non-coding intergenic rDNA spacer (IGS) to DNA finger print Giardia duodenalis isolates was investigated. Conserved PCR primers, specific for the flanking large and small rDNA genes, were used to amplify the IGS from 52 in vitro-cultured Giardia isolates. Four distinct IGS-PCR s ize groups (1.35-1.6 kb) were observed, which correlated closely with the m ajor genetic assemblages established previously for the same isolates using isoenzyme analysis. IGS-PCR size groups A (1.42 kb), C (1.4 kb) and D (1.3 5 kb) corresponded to isoenzyme assemblage A, and IGS-PCR group B (1.6 kb) to isoenzyme assemblage B. Amplified products from IGS-PCR size groups A an d B, which contained 50/52 isolates, were subsequently digested with 8 diff erent restriction enzymes and their profiles compared. Analysis separated i solates within each IGS-PCR size group into 2 distinct clusters which corre lated almost exactly with the same genetic groups established previously us ing isoenzyme electrophoresis. Within each cluster, both methods exhibited a similar capacity to distinguish between Giardia genotypes although they e stablished different genetic relationships between individual isolates. Muc h of the variability associated with the IGS was attributed to isolates har bouring multiple IGS-sequence types. Restriction analysis of IGS-PCR produc ts amplified from cloned and parent lines of a human isolate BAH 39, which contains multiple IGS variants, showed that trophozoite populations are hom ogeneous with respect to the types of IGS-variants they maintain. Furthermo re, in vitro culture of the cloned isolate BAH39c9 over a 6-year period als o failed to reveal variation in IGS-PCR digestion profiles. These results s uggest that IGS-PCR RFLP profiles are inherently stable. IGS-PCR analysis w as successfully applied to 11 Giardia cyst samples highlighting the potenti al for this approach to genotype Giardia isolates without the need for in v itro culture.