Abstrakt
This protocol describes an improved and optimized PCR-ELISA method for detection and quantification of Leishmania parasites in host tissues. unlike other DNA-based assays, this method uses digoxigenin-and biotin-labeled primers. this eliminates the need for a separate step of hybridization of the PCR product with labeled probes. the PCR product is detected using sandwich ELISA with antidigoxigenin-detecting antibodies. primers are complementary to the kinetoplast minicircle conserved region of parasite DNA, allowing the detection of several Leishmania species. For measurement of a wide range of parasite concentrations, +/- 25 cycles were optimal. the sensitivity of this technique is 0.3 fg of parasite DNA per reaction in 40-cycle PCR-ELISA, corresponding to 0.004 parasites.
DNA preparation by a standard TRI reagent procedure takes about 4 h. When DNA is prepared, a single person can test a large number of samples (at least 150) in a maximum of 7 h. this method might also be suitable for detecting and quantifying other pathogens, especially for detecting small differences in pathogen numbers