Leishmania parasite detection and quantification using PCR-ELISA
Language English Country England, Great Britain Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
20539283
DOI
10.1038/nprot.2010.68
PII: nprot.2010.68
Knihovny.cz E-resources
- MeSH
- DNA Primers genetics MeSH
- Enzyme-Linked Immunosorbent Assay methods MeSH
- Leishmania major genetics isolation & purification MeSH
- Leishmania genetics isolation & purification MeSH
- Leishmaniasis diagnosis parasitology MeSH
- Mice MeSH
- Polymerase Chain Reaction methods MeSH
- DNA, Protozoan analysis genetics MeSH
- Base Sequence MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA Primers MeSH
- DNA, Protozoan MeSH
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.
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