Differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii by a single-round PCR assay

Abstract

A single-round PCR assay was developed for detection and differential diagnosis of the three Entamoeba species found in humans, Entamoeba moshkovskii, Entamoeba histolytica, and Entamoeba dispar, that are morphologically identical as both cysts and trophozoites. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. PCR generates a 166-bp product with E. histolytica DNA, a 752-bp product with E. dispar DNA, and a 580-bp product with E. moshkovskii DNA. Thirty clinical specimens were examined, and the species present were successfully detected and differentiated using this assay. It was possible to detect as little as 10 pg of E. moshkovskii and E. histolytica DNA, while for E. dispar the sensitivity was about 20 pg of DNA. Testing with DNA from different pathogens, including bacteria and other protozoa, confirmed the high specificity of the assay. We propose the use of this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three morphologically indistinguishable Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys.

FIG. 1.

(A) Specificity of PCR with primers EntaF and combined primers EhR, EdR, and EmR in a single reaction mixture by using DNAs of various organisms extracted from pure or axenic cultures. Lane M, molecular weight marker (100-bp ladder); lane 1, E. histolytica DNA; lane 2, E. dispar DNA; lane 3, E. moshkovskii DNA; lane 4, human DNA; lane 5, Escherichia coli DNA; lane 6, Salmonella sp. DNA; lane 7, Shigella sp. DNA; lane 8, Vibrio cholerae DNA; lane 9, Blastocystis hominis DNA; lane 10, Giardia lamblia DNA; lane 11, Cryptosporidium sp. DNA; lane 12, DNA extracted from healthy human feces; lane 13, negative control. (B) Specificity of the designed primers by using DNA extracted from fecal samples containing various organisms. Lane M, molecular weight marker (100-bp ladder); lane 1, E. histolytica DNA; lane 2, E. dispar DNA; lane 3, E. moshkovskii DNA; lanes 4 and 5, Entamoeba coli DNA; lanes 6 and 7, Endolimax nana DNA; lanes 8 and 9, Giardia lamblia DNA; lanes 10 and 13, Cryptosporidium parvum; lane 11, E. histolytica; lane 12, E. dispar; lane 14, negative control.

FIG. 2.

Sensitivity of the PCR assay with twofold serial dilutions of E. histolytica DNA (A), E. dispar DNA (B), and E. moshkovskii DNA (C). Lane M, 100-bp ladder DNA marker; lane 1, 5 ng of DNA; lane 2, 2.5 ng of DNA; lane 3, 1.25 ng of DNA; lane 4, 0.625 ng of DNA; lane 5, 0.3125 ng of DNA; lane 6, 0.156 ng of DNA; lane 7, 0.078 ng of DNA; lane 8, 0.039 ng of DNA; lane 9, 0.019 ng of DNA; lane 10, 0.0095 ng of DNA; lane 11, 0.00475 ng of DNA; lane 12, 0.002375 ng of DNA; lane 13, 0.001188 ng of DNA; lane 14, 0.000594 ng of DNA.

FIG. 3.

PCR amplification for detection and differentiation of E. histolytica, E. dispar, and E. moshkovskii by using primers EntaF, EhR, EdR, and EmR. Lane M, 100-bp ladder DNA marker; lane 1, E. histolytica DNA; lane 2, E. dispar DNA; lane 3, E. moshkovskii DNA; lanes 4 to 7, amplified products (166 bp) which indicate the E. histolytica-positive specimens; lanes 8 to 13, amplified products (752 bp) which indicate the E. dispar-positive specimens; lane 14, negative control.