DNA Based Technologies - Methods of Detection of Parasites

DNA BASED TECHNOLOGIES

Nucleic acids, DNA or RNA, are found in all types of microorganism and the sequences of these molecules can be used to help in the identification of species and individuals. The robust nature of DNA has allowed researchers to use these sequence in a variety applications including forensics, archaeology (sequences from animal tissues over 100 years old have routinely been determined) and epidemiology. The advent of highly sensitive DNA amplification technologies such as the polymerase chain reaction (PCR) has allowed the development of molecular tools for the identification and detection of parasites. Initial work on these techniques focused on their ability to help define species and many of these studies utilized the ribosomal DNA genes and were very successful. Using these genes it was possible to discriminate many genera and species, but the genes lacked the level of sequence variation required for separation below the level of species (important for some pathogenic organisms and for epidemiological studies). In addition ribosomal DNA sequencing was also not always appropriate for identifying species that are highly diverse. Other DNA markers that have been used included genes that encode for metabolic enzymes and structural proteins. For example Cryptosporidium can be speciated by using a combination of target genes including the Cryptosporidium oocyst wall protein (COWP), heat shock protein (hsp70), dihydrofolate reductase (DHFR) and 18S ribosomal DNA. Complete genomes are now available for the major protozoan parasites and this has helped in the development of improved methods for species identification. These genomes can be accessed at http://eupathdb.org/eupathdb/ and this website links to many others resources including the National Centre for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/). Other DNAbased techniques are now available such as the multiplex PCR (amplification of more than one gene at a time) and quantitative real time PCR (qPCR), and these have improved specificity and sensitivity in detection and increased the usefulness of DNA technology for the detection of parasites in environmental as well as clinical samples. It is also possible to assess the viability of protozoan parasites using reverse transcription PCR (RTPCR). This is due to the fact that mRNAs degrade quickly once parasites are killed. However, it should be noted that increasing evidence exists to show that mRNA can survive in some cells for up to 24 hours

A recent method, DNA microarray, is a fast developing technology. This is a ‘chip’ based system that measures the binding of target DNA sequences (parasite) from samples (fluorescently labelled using PCR) to complementary sequences bound to the chip surface. The binding of parasite sequence DNA and the amount that is bound can be detected using fluorescence (Figure 6.9). In theory this technique allows the detection of multiple parasite species at the same time in one sample.