Identification of six Trypanosoma cruzi lineages by sequence-characterisedamplified region markers

Six discrete phylogenetic lineages were recently identified in Trypanosoma cruzi, on the basis of multilocus enzyme electrophoresis and random amplifi ed polymorphic DNA (RAPD) characterisation. The objective of the present st udy was to develop specific PCR-based markers for the identification of eac h of the six lineages. Eighty-seven T. cruzi stocks representative of all t he lineages were characterised by RAPD with three primers, resulting in the identification of three fragments that were specifically amplified in the given sets of lineages. After cloning and sequencing these fragments, three pairs of sequence-characterised amplified region (SCAR) primers were desig ned. After PCR amplification using the SCAR primers, the initial polymorphi sm was retained either as the presence or absence of amplification, or as s ize variation between the PCR products. Although most PCR products, taken i ndividually, were distributed across several lineages, the combination of t he three SCAR markers resulted in characteristic patterns that were distinc t in the six lineages. Furthermore, T. cruzi lineages were distinguished fr om Trypanosoma rangeli, T. cruzi marinkellei and T. cruzi-like organisms. T he excellent correspondence of these new PCR markers with the phylogenetic lineages, allied with their sensitivity, makes them reliable tools for line age identification and strain characterisation in T. cruzi. The approach de scribed here could be generalised to any species of microorganism harbourin g clear-cut phylogenetic subdivisions. (C) 2000 Elsevier Science B.V. Al ri ghts reserved.