Leishmania parasite detection and quantification using PCR-ELISA.
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.
Kobets
Tetyana
T
Laboratory of Molecular and Cellular Immunology, Institute of Molecular Genetics AS CR, v.v.i., Prague, Czech Republic.
Badalová
Jana
J
Grekov
Igor
I
Havelková
Helena
H
Svobodová
Milena
M
Lipoldová
Marie
M
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007891:Leishmania / Q000235:genetics* / Q000302:isolation & purification*; D018320:Leishmania major / Q000235:genetics / Q000302:isolation & purification; D007896:Leishmaniasis / Q000175:diagnosis / Q000469:parasitology; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D016054:DNA, Protozoan