[Last Modified: ]
[Entamoeba histolytica]

Molecular diagnosis

Conventional PCR

A

A: Agarose gel (2%) analysis of a PCR diagnostic test for differentiation between E. histolytica and E. dispar.

• Lanes 1 - 4: Amplification with the Psp3/Psp5¹ PCR primer pair specific for E. histolytica.  Diagnostic band size - 876 bp.
• Lanes 6 - 9: Amplification with the NPsp3/NPsp5¹ PCR primer pair specific for E. dispar.  Diagnostic band size - 876 bp.
• Lanes 1 and 6: E. histolytica 200:NIH, zymodeme II (positive control for E. histolytica).
• Lanes 2 and 7: E. dispar 351:IMMiT, zymodeme I (positive control for E. dispar).
• Lanes 3 and 8: Specimen from a patient with a liver abscess (positive with E. histolytica primers and negative with E. dispar primers).  E. histolytica 333:IMMiT, zymodeme XIV.
• Lanes 4 and 9: Specimen from an asymptomatic patient (positive with E. dispar primers and negative with with E. histolytica primers).  E. dispar 389:IMMiT, zymodeme I.
• Lane 5: Molecular base pair standard, 100-bp ladder (from 600 to 1,000 bp).

Figure contributed by Assist. Prof. Przemyslaw Myjak, Ph.D., Institute of Maritime and Tropical Medicine, Gdynia, Poland.

Real-Time PCR

B

B: The principle of TaqMan real-time PCR is depicted in the schematic above².  The TaqMan probe has a reporter dye at it 5́  end and a quencher dye at its 3́  end.  (1) No fluorescent signal is emitted during the denaturation step even after excitation by the light source of the instrument, because the intact TaqMan probe has both reporter and quencher fluorochromes in close proximity.  The energy absorbed by the reporter is transferred to the nearby fluorochrome, in which case it results in the quenching of the fluorescence (black arrow in 1, 2, and 3).  The probe is designed to be complementary to a specific sequence spanned by the PCR primers.  Usually, the 5́  end of the probe hybridizes to a few nucleotides bases after the 3́  end of the forward or reverse PCR primer.  The TaqMan probe hybridizes to the DNA template during the phase that the temperature is lowered to allow annealing of the PCR primers (2); no fluorescence is produced because the probe is still intact and the dyes are in close proximity.  After the annealing (3), the PCR primers are extended and the DNA polymerase cleaves the TaqMan probe, releasing the reporter from the proximity of the quencher (3 and 4).  The reporter dye, after excitation by the light source from the instrument, emits a fluorescent signal.  Amplification of DNA is tracked by accumulation of fluorescent signal produced in each PCR cycle.  A TaqMan real-time PCR approach has been validated at CDC and is used for differential laboratory diagnosis of amebiasis.^{3, 4}

References

1. Clark CG, Diamond LS. Differentiation of pathogenic Entamoeba histolytica from other intestinal protozoa by riboprinting. Arch Med Res 1992;23:15-16.
2. da Silva A, Pieniazek N. Latest Advances and Trends in PCR-based Diagnostic Methods. In: Dionisio D, editor. Textbook-Atlas of Intestinal Infections in AIDS. Springer; 2003. p. 397-412.
3. Qvarnstrom Y, James C, Xayavong M, Holloway B, Moura I, Visvesvara GS, et al. Comparison of real-time PCR rationales for differential laboratory diagnosis of amebiasis. J Clin Microbiol 2005;43:3014-3016.
4. Verweij JJ, Vermer J, Brienen EA, Blotkamp C, Laeijendecker D, van Leishout L, Polderman AM. Entamoeba histolytica infections in captive primates. Parasitol Res 2003;90:100-3.