Standardization of molecular techniques for the detection and characterization of intestinal protozoa and other pathogens in humans.

Background: The intrinsic sensitivity limitations of basic parasitological methods, along with the particular biological characteristics of parasites, make these methods ineffective to differentiate morphologically indistinguishable species. Molecular detection and characterization techniques could be used to overcome these problems. The purpose of this work was to standardize molecular polymerase chain reaction (PCR) techniques, described in the literature, for the detection and molecular characterization of intestinal protozoa and other pathogens in humans. Methods: DNA was extracted from human or animal feces, previously washed or cultured in Boeck Drbohlav's Modified Medium. DNA extraction was performed with Machery-Nagel extraction kits. The standardization of the PCR, nested-PCR or RFLP techniques was carried out according to the literature. For each molecular technique performed, the sensitivity of the test was determined based on the minimun quantity required of DNA (sensitivity A) and the minimum quantity of life forms that the test detected (sensitivity B). Results: Sensitivity A was 10 fg for Conclusions: The molecular detection of protozoa and chromist was achieved and the molecular characterization allowed the genotyping of some of the parasites such as