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Leishmaniasis

Leishmaniasis

Leishmaniasis is a protozoal disease caused by species of the genus Leishmania and transmitted through the bite of infected female sandflies. The parasite exists in two principal morphological forms during its life cycle. A sandfly acquires intracellular amastigotes from an infected reservoir host, such as a dog. Within the sandfly, these forms differentiate into motile, flagellated promastigotes. During a subsequent blood meal, promastigotes are injected into the human host, where they...

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Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples

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Leishmania parasite detection and quantification using PCR-ELISA.

Tetyana Kobets¹, Jana Badalová, Igor Grekov

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

Nature Protocols

|June 12, 2010

Summary

This study presents an optimized PCR-ELISA technique for detecting Leishmania parasites. The enhanced method uses labeled primers, simplifying detection and improving quantification of parasite DNA in host tissues.

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Area of Science:

Molecular Biology
Parasitology
Immunology

Background:

Leishmania parasites cause significant global health issues.
Accurate detection and quantification of Leishmania DNA are crucial for diagnosis and treatment monitoring.
Existing DNA-based assays can be complex and time-consuming.

Purpose of the Study:

To describe an improved and optimized Polymerase Chain Reaction-Enzyme Linked Immunosorbent Assay (PCR-ELISA) for Leishmania detection.
To enhance the efficiency and sensitivity of parasite DNA quantification in host tissues.
To provide a streamlined method for detecting multiple Leishmania species.

Main Methods:

Utilized digoxigenin- and biotin-labeled primers for direct PCR product detection, eliminating hybridization steps.
Employed a sandwich ELISA format with antidigoxigenin-detecting antibodies for detection.
Primers targeted the conserved kinetoplast minicircle region of Leishmania DNA.
Optimized PCR cycling to +/-25 cycles for broad concentration measurement.

Main Results:

Achieved a sensitivity of 0.3 fg of parasite DNA per reaction with 40-cycle PCR-ELISA, equivalent to 0.004 parasites.
Demonstrated the ability to detect multiple Leishmania species.
The protocol allows for high-throughput sample processing (≥150 samples/7 hours) after DNA preparation (4 hours).

Conclusions:

The optimized PCR-ELISA offers a sensitive, specific, and efficient method for Leishmania parasite detection and quantification.
This streamlined assay simplifies parasite DNA detection in clinical and research settings.
The method's adaptability suggests potential for detecting and quantifying other pathogens.