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Journal of Clinical Microbiology, May 2002, p. 1666-1669, Vol. 40, No. 5
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.5.1666-1669.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Real-Time PCR for Detection and Quantitation of Leishmania in Mouse Tissues

Luc Nicolas,* Eric Prina, Thierry Lang, and Geneviève Milon

Unité d'Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, 75724 Paris Cedex 15, France

Received 15 November 2001/ Returned for modification 20 January 2002/ Accepted 25 February 2002

Leishmania spp. are intracellular protozoan parasites that cause a wide spectrum of diseases in humans and dogs worldwide. However, monitoring of the Leishmania burden in its different hosts is still based on cumbersome and poorly sensitive methods. Here we have developed a highly accurate real-time PCR assay with which to reproducibly detect and quantify the relative Leishmania major burden in mouse tissue samples. The assay is performed with the LightCycler system using SYBR Green I and primers amplifying a ca. 120-bp fragment from minicircles of the kinetoplast DNA (kDNA). The assay was able to detect as little as 100 fg of L. major DNA per reaction, which is equivalent to 0.1 parasite. The standard curve designed for quantitation of parasites showed linearity over an at least 6-log DNA concentration range, corresponding to 0.1 to 104 parasites per reaction, with a correlation coefficient of 0.979. The assay also proved to have a detection range of the same magnitude as that used for detection of L. donovani and L. amazonensis, but it was 100-fold less sensitive for L. mexicana. When applied to tissues from experimentally infected mice, the real-time PCR assay is not only as sensitive as a conventional PCR assay for detection of Leishmania kDNA but also more rapid. Results indicate that this assay is compatible with the clinical diagnosis of leishmaniasis and will be a great help to scientists who use animals to monitor the efficacy of antileishmanial drugs or vaccines or decipher the unique properties of the life cycle of Leishmania spp .

* Corresponding author. Mailing address: Institut Pasteur, Unité d'Immunophysiologie et Parasitisme Intracellulaire, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: (33) 1 45 68 81 70. Fax: (33) 1 40 61 31 69. E-mail: lnicolas{at}pasteur.fr.

Journal of Clinical Microbiology, May 2002, p. 1666-1669, Vol. 40, No. 5
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.5.1666-1669.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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