Development of nested PCR for identification of Entamoeba coli in stool samples

Abstract Background The accurate detection of Entamoeba species provides for a better understanding of the nature of these parasites in humans and helps to provide accurate epidemiological data. However, the microscopic method is not sensitive and specific to identify the precise identification of Entamoeba spp. in stool specimens especially if it was examined by inexperienced laboratory technicians. In this study, nested PCR was developed for detection of Entamoeba coli , and can be differentiated from the other human Entamoeba spp. especially Entamoeba histolytica, Entamoeba dispar , and Entamoeba moshkovskii . Methods Nested PCR assay was performed using Entamoeba genus-specific primers for the first round PCR reaction and newly designed E. coli -specific primers targeting small subunit ribosomal RNA (SSU rRNA) gene in the second round. Both sensitivity and specificity of nested PCR for detection of E. coli were determined. This assay was validated for detection of E. coli by using DNA obtained from 55 stool samples which previously examined by microscopy. Results obtained from developed nested PCR, genus-specific PCR, and microscopy were analyzed. Results Nested PCR was successfully developed to diagnose E. coli infection with a product size of 166 base pairs (bp). The nested PCR method could detect as little as 1 pg of E. coli DNA. The high specificity was confirmed by testing with DNA from the different intestinal pathogens, including bacteria and other protozoa. After testing with all stool samples, 50.9% of tested samples were positive for Entamoeba spp. by genus-specific PCR assay, 32.7% of E. coli positive samples detected by nested PCR, and 29.1% were positive by microscopic examination. Interestingly, four samples previously negative for E. coli by microscopy demonstrated E. coli by nested PCR. In addition, microscopy showed two false-positive compared with both nested PCR and genus-specific primers results. Conclusions The developed nested PCR assay based on genus Entamoeba -specific primers and newly designed E. coli -specific primers shows high sensitivity and specificity for the diagnosis of E. coli in stool samples. This nested PCR assay will be beneficial not only for diagnostic purposes but also for a better understanding of the epidemiology of E. coli in the human population worldwide.