Detection and genotyping of Giardia intestinalis isolates using intergenic apacer (IGS)-based PCR

Abstract

Giardia intestinalis infections arise primarily from contaminated food or water. Zoonotic transmission is possible, and at least 7 major assemblages including 2 assemblages recovered from humans have been identified. The determination of the genotype of G. intestinalis is useful not only for assessing the correlation of clinical symptoms and genotypes, but also for finding the infection route and its causative agent in epidemiological studies. In this study, methods to identify the genotypes more specifically than the known 2 genotypes recovered from humans have been developed using the intergenic spacer (IGS) region of rDNA. The IGS region contains varying sequences and is thus suitable for comparing isolates once they are classified as the same strain. Genomic DNA was extracted from cysts isolated from the feces of 5 Chinese, 2 Laotians and 2 Koreans infected with G. intestinalis and the trophozoites of WB, K1, and GS strains cultured in the laboratory, respectively. The rDNA containing the IGS region was amplified by PCR and cloned. The nucleotide sequence of the 3' end of IGS region was determined and examined by multiple alignment and phylogenetic analysis. Based on the nucleotide sequence of the IGS region, 13 G. intestinalis isolates were classified to assemblages A and B, and assemblage A was subdivided into A1 and A2. Then, the primers specific to each assemblage were designed, and PCR was performed using those primers. It detected as little as 10 pg of DNA, and the PCR amplified products with the specific length to each assemblage (A1, 176 bp; A2, 261 bp; B, 319 bp) were found. The PCR specific to 3 assemblages of G. intestinalis did not react with other bacteria or protozoans, and it did not react with G. intestinalis isolates obtained from dogs and rats. It was thus confirmed that by applying this PCR method amplifying the IGS region, the detection of G. intestinalis and its genotyping can be determined simultaneously.