Počet záznamů: 1

Leishmania parasite detection and quantification using PCR-ELISA

  1. 1.
    0355298 - UMG-J 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
    Grant CEP: GA ČR GA310/08/1697; GA MŠk(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
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