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Nat Protoc. 2010 Jun;5(6):1074-80. doi: 10.1038/nprot.2010.68. Epub 2010 May 20.

Leishmania parasite detection and quantification using PCR-ELISA.

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1
Laboratory of Molecular and Cellular Immunology, Institute of Molecular Genetics AS CR, v.v.i., Prague, Czech Republic.

Abstract

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.

PMID:
20539283
DOI:
10.1038/nprot.2010.68
[Indexed for MEDLINE]
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