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Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples.

ABSTRACT: Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved region of the 18S ribosomal RNA (rRNA) gene; amplification was visualized by the pre-amplification addition of fluorescent detection reagent (FDR) and a simple UV lamp. By using a reverse-transcriptase step, the system detected infections between 10 and 100 parasites per mL. The assay was tested on a range of nucleic acid extracts from Leishmania species, visceral leishmaniasis (VL) patients from Sudan, and cutaneous leishmaniasis (CL) patients from Suriname. The sensitivity of RT-LAMP from the blood of VL patients was 83% (N = 30) compared with microscopy of bone-marrow and lymph-node aspirates; for CL patients the observed sensitivity was 98% (N = 43). The potential to use LAMP as a diagnostic tool for leishmaniasis is discussed.

SUBMITTER: Adams ER

PROVIDER: S-EPMC2844582 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples.

Adams Emily R ER Schoone Gerard J GJ Ageed Al Farazdag AF Safi Sayda El SE Schallig Henk D F H HD

The American journal of tropical medicine and hygiene 20100401 4

Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved region of the 18S ribosomal RNA (rRNA) gene; amplification was visualized by the pre-amplification addition of fluorescent detection reagent (FDR) and a simple UV lamp. By using a reverse-transcriptase ...[more]

PMID: 20348505

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Project description:Leishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genus Leishmania. Recently, an increasing number of autochthonous leishmaniasis cases caused by L. martiniquensis and the novel species L. siamensis have been described in Thailand, rendering an accurate diagnosis of this disease critical. However, only a few laboratories are capable of diagnosing leishmaniasis in Thailand. To expand leishmaniasis diagnostic capabilities, we developed a simple colorimetric loop-mediated isothermal amplification (LAMP) technique for the direct detection of Leishmania DNA.LAMP was performed for 75 min using four primers targeting the conserved region of the18S ribosomal RNA gene, and the DNA indicator used was malachite green (MG). To simulate crude samples, cultured promastigotes of L. siamensis were mixed with blood or saliva. Also, clinical samples (blood, saliva, and tissue biopsies) were obtained from patients with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). All samples were boiled for 10 min and introduced directly into the LAMP reaction mixture without DNA purification.The use of MG resulted in an unambiguous differentiation of positive and negative controls. For L. siamensis, the detection limit was 10(3) parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of active Leishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multiple Leishmania species other than L. siamensis and L. martiniquensis, including L. aethiopica, L. braziliensis, L. donovani and L. tropica.The simplicity and sensitivity of LAMP in detecting active Leishmania infection could enable the rapid diagnosis of leishmaniasis, thereby facilitating the survey and control of leishmaniasis in Thailand. However, our limited number of samples warranted a further validation with a larger cohort of patients before this assay could be deployed.

Dataset Information

Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples.

Publications

Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples.

Similar Datasets

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