from Theron et al. in Nanotechnology in Water Treatment Applications

Detection of pathogenic organisms provides information as to the safety and public health risks associated with a given water supply; however, it often does little to define the potential sources of the contamination. Generally, because different enteric pathogens are present in the intestines of different animals, the identification of a contamination event as being either of human or animal source would provide information as to the types of pathogens that may be expected, the risk of infection and the treatment that may be required to control transmission of disease. In response, molecular techniques are being developed as means to identify the source of a contaminant. DNA fingerprinting is one tool for microbial source tracking (MST) and consists of a family of techniques that are used to identify the sources of fecal contamination in various water bodies.

The different polymorphism-based procedures are generally coupled to a PCR reaction. In the amplified ribosomal DNA restriction analysis (ARDRA) technology, PCR-amplified 16S rRNA genes are digested with restriction endonucleases and the resulting fragments separated electrophoretically. Presence or absence of the restriction site within two strains cause differences in the length of the DNA restriction fragments, and the complexity of the pattern depends upon the number of target sequences and position of restriction sites. Comparison of the generated patterns to those obtained from a database allows assignment of isolates to species or species clusters in those cases were the banding patterns are highly similar. The separated DNA fragments may also be transferred to filters for hybridization with probes specific for an organism of interest. Two other protocols for generating DNA fingerprints use a single primer to amplify fragments with PCR before examination on agarose gels. PCR amplification of repetitive extragenic palindromic sequences (Rep-PCR) takes advantage of repetitive sequences found in the microbial genome. In the randomly amplified polymorphic DNA (RAPD) or arbitrarily primed PCR technology, a short oligonucleotide primer (about 10 nucleotides), usually with random sequence that is not specific for a particular gene is used as a primer to amplify fragments. These methods yield DNA fingerprints comprised of multiple, differently sized DNA amplification products following separation by gel electrophoresis.

Detection of host-specific 16S rRNA genetic markers, using length heterogeneity PCR (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) analysis, also holds promise as an effective method for characterizing a microbial population. The technique distinguishes members of mixtures of bacterial gene sequences by detecting differences in the number of base pairs in a particular gene fragment. Whereas LH-PCR separates PCR products for host-specific genetic markers based on the length of amplicons, T-RFLP uses restriction enzymes on amplified PCR products to determine unique size fragments. Specifically, in T-RFLP, rRNA target gene sequences are PCR-amplified using one or both of the primers with a fluorescent label. The amplification product(s) are then digested with appropriate restriction endonucleases and following electrophoresis of the resultant fragments using an automated DNA sequencer, a fluorescent electrophoretic profile of the digestion patterns is obtained. The use of labelled primers limits the analysis (identification) to only the terminal fragments, thus allowing the study of complex microbial communities. Moreover, the possibility of discriminating fragments with differences as small as single bases gives the method a higher resolution than gel-based profiling techniques.

One of the most promising technologies for microbial source tracking is, however, amplified fragment length polymorphism (AFLP) analysis. AFLP analysis appears to have the same taxonomic range as other fingerprinting techniques, but this technology combines several advantages of these different techniques, which in most cases results in the highest power of discrimination. This technology is based on the selective amplification of a subset of genomic restriction fragments using PCR. For AFLP, purified genomic DNA is digested with two restriction endonucleases, one with an average cutting frequency and a second one with a higher cutting frequency after which oligonucleotide adapters are ligated to the genomic DNA restriction fragments. The sequence of the adapters and the adjacent restriction site serves as oligonucleotide primer binding sites for subsequent amplification of the restriction fragments by PCR. Selective nucleotides extending into the restriction fragments are added to the 3' ends of the adapter-specific PCR primers such that only a subset of the restriction fragments are recognized and amplified. The subset of amplified fragments is then analyzed by denaturing polyacrylamide gel electrophoresis to generate the fingerprint. Since relatively small amounts of DNA are digested and detection of AFLP fragments does not depend on hybridization, the AFLP analysis method is more reproducible and robust than other fingerprinting techniques and it also displays more fragments than other fingerprinting techniques.

Tags: Microbial Detection | Pathogen Detection | Biodiagnostics | Biodetection Assays | Biomolecular Detection