PCR based approaches to the identification and classification of Leishmania

A. Noyes, H. (1996) PCR based approaches to the identification and classification of Leishmania. PhD thesis, University of Liverpool.

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Abstract

Random amplified polymorphic DNA (RAPD) was tested for the identification and classification of Leishmania. RAPD was found to be useful for the identification of species of L. (Leishmania) and L. (Yiannia) and for the classification of L. (Yiannia) species. The polymerase chain reaction (PCR) was tested for the identification of Leishmania from mammals and lizards, using both published primers and new primers which amplify kinetoplast minicircle DNA. The size of the PCR product was found to be useful for discriminating between some sympatric pairs of species such as L. braziliensis and L. mexicana. Isotopically labelled probes prepared from the variable region of the kinetoplast minicircle were tested for specificity for the identification of New and Old World species of Leishmania. The specificity was dependent on the concentration of target DNA and was manipulated to investigate relationships between Leishmania species. Restriction digests of kinetoplast DNA (schizodemes) prepared by PCR and by centrifugation through 20% sucrose were compared for the identification of strains of L. infantum and L. chagasi. Twenty three strains of L. chagasi from cases of visceral and cutaneous leishmaniasis in Honduras were examined by RAPD, schizodemes, differential display, isoenzyrnes, RFLPs and PFGE to discover whether genetic differences existed between parasites causing the two different pathologies. The parasites were found to be unusually homogeneous and no differences were found which correlated with pathology by any of these methods. Restriction digests of PCR amplified small subunit ribosomal DNA (SSU rDNA) (ribodemes) were tested to find markers specific for the genus Leishmania. A classification of the Leishmania based on the restriction fragments indicated that L. hertigi and L. herreri were more closely related to Endotrypanum than to Leishmania, and that the lizard Leishmania could not be placed in separate genus from the Leishmania. Ribodemes were used to identify two strains of parasites supplied by colleagues in Central America that could not be identified by existing methods for the identification of Leishmania. One of these strains appeared to be identical to a C. luciliae reference strain. The other strain produced a fingerprint unlike any of the available reference strains. A variable region of the SSU rRNA gene was identified that was suitable for classifying trypanosomatids and the sequence of this region was used to classify the strain that could not be identified by fingerprinting.

Item Type:	Thesis (PhD)
Depositing User:	Symplectic Admin
Date Deposited:	23 Oct 2023 09:57
Last Modified:	23 Oct 2023 09:57
DOI:	10.17638/03175946
Copyright Statement:	Copyright © and Moral Rights for this thesis and any accompanying data (where applicable) are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge.
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3175946