Molecular approaches to DNA diagnosis

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Abstract
SUMMARY: The DNA of a parasite is the ultimate blueprint of that parasite, the one characteristic which normally remains unchanged during every stage of the life-cycle. All the DNA sequence in the egg of a species of parasite are also in the larvae and adults of the same species. The same DNA is present in the parasite whether it is in a free-living stage, in an invertebrate vector or in a vertebrate host such as man. The molecular basis for DNA diagnosis is to allow labelled single-stranded species or strain-specific DNA sequences, selected from well-characterized reference species, to find and hybridize with homologous DNA from, or in, the unknown isolates of parasites. DNA probes are now available for most vector borne parasitic diseases. Parasitological identification problems are mostly concerned with distinguishing closely related strains or subspecies, for example detectingTaenia soliumeggs as opposed toT. saginataeggs, or finding which of the 15 man-infecting subspecies ofLeishmaniais present in a single cutaneous lesion, the commonest clinical sign of the disease, or in a sandfly. For efficient hybridization by the present methods there has to be enough of a particular sequence present in a parasite's genome to make a feasible target. Therefore, DNA probes for parasites have been selected from repetitive, reiterated or multicopy DNA with intrinsic extensive sequence variation. DNA, which is free of coding restraint, can evolve rapidly to give differences between species, so that introns, ribosome gene spacers, variant genes, pseudo-genes and non-conserved DNA have all been used for DNA diagnosis. The major problems of sequence selection have been greatly aided by the use of recombinant DNA methods, which have the added advantage of economical production of DNA probes. The unique characteristics of kinetoplast mini-circle DNA inLeishmaniahas allowed the selection of a complex species, subspecies, strain and even isolate-specific DNA probes. These have been used successfully for Southern filter endonuclease fragment DNA identification, for dot-blot recognition of less than 200 parasites and non-radioactive detection of DNA sequence homology by ‘in situ’ hybridization and light microscopy in a singleLeishmaniacell. The adaptation of the forensic human genetic fingerprinting technique has allowed identification ofL. braziliensisDNA in human biopsy material, even the presence of a vast excess of human DNA. Fingerprinting with ribosomal spacer-derived recombinant DNA probes has been used to discriminateEchinococcusspecies. The identification ofTaeniaspecies has been accomplished using probes from a genomic library of size-selected DNA fragments, and synthetic oligonucleotides are available forOnchocercasubspecies detection. The new technologies combining repeated genomic sequence probes with pulse field separation of the chromosomes of parasites, has opened up new avenues of research. Double probes for the simultaneous identification of the insect vector and the carried parasite have recently been reported. Lastly, the polymerase chain reaction technique has given us the opportunity of amplifying even single copy genes in one parasite to give sufficient DNA for positive identification. DNA diagnosis in parasitology is now on a par with the DNA diagnosis of viruses and human genetic disorders. The last ten years have seen many exciting developments in DNA diagnosis; the next years should see DNA diagnosis routinely used in epidemiological and clinical situations in countries where parasitic diseases are a major public health problem.