Leishmania parasite detection and quantification using PCR-ELISA

0355298 - ÚMG 2011 RIV GB eng J - Článek v odborném periodiku
Kobets, Tetyana - Badalová, Jana - Grekov, Igor - Havelková, Helena - Lipoldová, Marie
Leishmania parasite detection and quantification using PCR-ELISA.
Nature Protocols. Roč. 5, č. 6 (2010), s. 1074-1080. ISSN 1754-2189. E-ISSN 1750-2799
Grant CEP: GA ČR GA310/08/1697; GA MŠMT(CZ) LC06009
Výzkumný záměr: CEZ:AV0Z50520514
Klíčová slova: polymerase chain reaction * Leishmania major infection * parasite quantification
Kód oboru RIV: EB - Genetika a molekulární biologie
Impakt faktor: 8.362, rok: 2010

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.
Trvalý link: http://hdl.handle.net/11104/0194106