Title: Rapid sequencing of rDNA from single worms and eggs of parasitic helminths
Abstract: The development of highly sensitive diagnostic techniques for the accurate identification of individual eggs of parasite species of medical and veterinary importance is central to the control of the diseases they cause.Sequencing of ribosomal genes provides a powerful molecular tool for species-level diagnosis and phylogenetic studies (1-3), and is usually based on either of the two original techniques (4, 5).Direct PCR cycle sequencing (6, 7) is an attractive approach, because it is rapid, labour effective, and can be used to generate template from minute quantities of material (8,9).This is especially important since often only limited specimens are available.This method also enables DNA template to be sequenced at high temperatures, thus reducing artifacts due to local secondary structure.Sequencing techniques rely on pure template DNA.Unfortunately, it is often difficult to isolate sufficient and pure DNA template from some parasitic helminths, because of their tough cuticle (10) and a 'white flocculate' substance found to co-precipitate with DNA during isolation (11,12), which inhibits subsequent enzymatic reactions.In this paper, we describe a DNA isolation method which overcomes these problems and a PCR cycle sequencing technique which is sufficiently sensitive to sequence rDNA from single nematode eggs.