Use of PCR amplification of tRNA gene-linked short tandem repeats for genotyping Entamoeba histolytica

Abstract

We have developed a reliable method for PCR-based genotyping of Entamoeba histolytica based on variation in the numbers of short tandem repeats that are linked to tRNA genes in this species. Species-specific primer pairs were designed that differentiate E. histolytica from E. dispar as well as that reveal intraspecies PCR product length polymorphisms. The primers were tested with samples from different parts of the world, and DNA was extracted from cultured cells as well as liver abscess pus and feces by various methods. We now have the tools necessary to investigate a possible link between parasite genotype and the outcome of infection with Entamoeba histolytica, as well as other aspects of the organism's epidemiology.

FIG. 1.

Examples of polymorphism observed in tRNA-linked STRs of E. histolytica. (A) Three STRs showing little or no PCR product size polymorphism; (B) three of the selected STRs showing low to moderate polymorphisms; (C) the remaining three selected STRs showing moderate to high polymorphisms. All samples except E. dispar SAW760 are E. histolytica.

FIG. 2.

STR polymorphisms obtained by using different sample types. (A) STR polymorphisms obtained by using DNA from liver abscess samples (lanes LAN-15 and LAN-39) and E. histolytica xenic culture DNAs (other lanes) at two of the selected STRs. The tRNA-specific primers were used (Table 2). (B) STR polymorphisms obtained by using liver abscess (LAID-19 and LAID-31) and fecal DNAs. Lanes 22027 and 29621, fecal DNAs from Bangladesh isolated by a modified CTAB method; lanes 231-1/4.00 and 484-1/4.01, fecal DNAs from Vietnam isolated by using the QIAamp DNA stool mini kit. The E. histolytica species-specific primers (Table 2) were used.

FIG. 3.

Species-specific primer testing. Each of the DNA samples was amplified by using both the E. histolytica-specific (H) and the E. dispar-specific (D) primers for two of the selected STRs, S^TGA-D and S-Q (Table 2). E. histolytica HM-1:IMSS and E. dispar SAW760 were used as controls. Strains 31 and 32 are known E. histolytica isolates, while strains 121 and 122 are known E. dispar isolates from Bangladesh, and the DNA was extracted from xenic cultures.