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Leishmania parasite detection and quantification using PCR-ELISA

Nature Protocols volume 5, pages 1074–1080 (2010)Cite this article

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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.

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Figure 1: PCR-ELISA scheme.
Figure 2: Sensitivity of detection by PCR.
Figure 3: Standard curves.
Figure 4: Giemsa-stained lymph node smears.
Figure 5: Parasite load measured with two different techniques.

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Acknowledgements

This investigation was supported by the Grant Agency of the Czech Republic (Grant no. 310/08/1697), by the Academy of Sciences of the Czech Republic (Grant no. AVOZ50520514) and by the Ministry of Education of the Czech Republic (LC06009). We thank Dr Eva Nohýnková (First Faculty of Medicine, Charles University, Prague, Czech Republic) for technical advice.

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Authors and Affiliations

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

    Tetyana Kobets, Jana Badalová, Igor Grekov, Helena Havelková & Marie Lipoldová

  2. Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic

    Milena Svobodová

Authors
  1. Tetyana Kobets
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  2. Jana Badalová
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  3. Igor Grekov
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  4. Helena Havelková
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  5. Milena Svobodová
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  6. Marie Lipoldová
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Contributions

T.K., J.B. and M.L. conceived and designed the method; T.K., H.H. and I.G. extended and refined the method; M.S. performed parasitology experiments; and T.K., J.B. and M.L. wrote this paper.

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Correspondence to Marie Lipoldová.

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The authors declare no competing financial interests.

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Kobets, T., Badalová, J., Grekov, I. et al. Leishmania parasite detection and quantification using PCR-ELISA. Nat Protoc 5, 1074–1080 (2010). https://doi.org/10.1038/nprot.2010.68

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