Number of the records: 1
Leishmania parasite detection and quantification using PCR-ELISA
- 1.
SYSNO ASEP 0355298 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Leishmania parasite detection and quantification using PCR-ELISA Author(s) Kobets, Tetyana (UMG-J) RID
Badalová, Jana (UMG-J)
Grekov, Igor (UMG-J) RID
Havelková, Helena (UMG-J)
Lipoldová, Marie (UMG-J) RIDSource Title Nature Protocols. - : Nature Publishing Group - ISSN 1754-2189
Roč. 5, č. 6 (2010), s. 1074-1080Number of pages 7 s. Language eng - English Country GB - United Kingdom Keywords polymerase chain reaction ; Leishmania major infection ; parasite quantification Subject RIV EB - Genetics ; Molecular Biology R&D Projects GA310/08/1697 GA ČR - Czech Science Foundation (CSF) LC06009 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) CEZ AV0Z50520514 - UMG-J (2005-2011) UT WOS 000278354700010 DOI 10.1038/nprot.2010.68 Annotation 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. Workplace Institute of Molecular Genetics Contact Nikol Škňouřilová, nikol.sknourilova@img.cas.cz, Tel.: 241 063 217 Year of Publishing 2011
Number of the records: 1