A modified PCR-RFLP method to determine genetic diversity of Giardia lamblia human isolates based on triosephosphate isomerase (TPI) gene

Abstract

An infection of digestive system, Giardiasis, caused by tiny parasites called Giardia lamblia (also known Giardia intestinalis or Giardia duodenalis). Giardia sp. is the most common intestinal parasite of humans and other animals throughout the world. Isolates of G. lamblia are classified into eight assemblages based on isoenzyme and DNA analyses. Assemblages A and B infect humans and a broad range of other hosts. The purpose of this study was to genotype human isolates of G. lamblia by PCR-RFLP in Karaj city. 60 positive fecal samples of G. lamblia were collected. DNA extraction and amplification of TPI gene were successfully conducted by nested-PCR. Subsequently, all samples were positive. Sequencing on 5 samples was conducted to determine genetic differences. The presence of 2 genotypes of G. lamblia (A and B) was revealed by the alignment of the TPI sequences obtained with reference sequences. The results of RFLP technique show that 35 of 60 (58.3%) isolates belonged to assemblage A, and 17 of 60 (28.3%) belonged to assemblage B but 1(1.7%) sample was not determined. Whereas, 7 (11.6%) specimens were detected as mixed infections. The latter RFLP was carried out to identify subtypes.The final results were 100% (35/35) AII, 82.3% (14/17) BIII, and 17.7% (3/17) BIV. This study suggests that the modified RFLP method is favorably time saving and easily achievable and highly economical. Hence, the sub-assemblage AII might be dominant in Karaj city.

Keywords: Genotype; Giardia lamblia; Triose phosphate isomerase gene.