Leishmania parasite detection and quantification sing PCR-ELISA

This protocol describes an improved and optimized PCR -EL ISA 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 EL ISA 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 -EL ISA , corresponding to 0.004 parasites. DNA preparation by a standard TR I 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 di fferences in pathogen numbers.