from Theron et al. in Nanotechnology in Water Treatment Applications

Nucleic acid hybridization techniques
The easiest way of detecting specific nucleic acid sequences is through direct hybridization of a probe to microbial nucleic acid extracts. These hybridization techniques rely on the specific binding of nucleic acid probes to complementary DNA or RNA (target nucleic acid). The probes are single strands of nucleic acid with the potential of carrying detectable marker molecules highly specific to complementary target sequences, even if these sequences account for only a small fraction of the target nucleic acid. Either DNA or RNA can serve as a nucleic acid probe, but for a number of reasons (e.g., ease of synthesis and stability), most studies have employed DNA probes. The probes may be used to detect genes in the bacterial genome (Southern blots) or to detect mRNA or rRNA (Northern blots).

According to their length, DNA probes can be grouped as either polynucleotide probes (more than 50 nucleotides) or as oligonucleotide probes (less than 20 nucleotides). The latter are more frequently used since they are short enough to allow for single mismatch discrimination of target nucleic acids and large quantities of oligonucleotides can be rapidly and inexpensively produced. During oligonucleotide synthesis a variety of marker or linker sequences can be introduced to the 5' end of the oligonucleotide. The principle conjugate fluorochromes are derivatives of fluorescein or rhodamine, although labels such as digoxigenin and biotin have also been used. For probes to be effective in nucleic acid hybridization, they must be specific and selective. This requires that sequence data be available. Although such data is available for some waterborne pathogens, the data are not comprehensive for all pathogens of concern. However, the use of bacterial 16S rRNA sequences to develop determinative hybridization probes is now well established. The occurrence of 16S rRNA molecules in high copy numbers of usually more than 1000 in any living cell, as well as the fact that 16S rRNA sequences have been determined for a large fraction of bacterial species, currently confers on these genes a high informative value at phylogenetic level. Comparative analyses of rRNA sequences have indicated that the rRNA molecules comprise highly conserved sequence domains interspersed with more variable regions. Consequently, so-called signature sequence motifs on various taxonomic levels, from domains to subspecies, can be identified. Whereas species-specific probes complement the most variable regions, more general probes target more conserved regions of the molecule. Although several oligonucleotide probes are commercially available, new specific probes may also be designed. The design of such 16S rRNA-targeted probes is greatly facilitated by computer-aided sequence comparisons of large rRNA sequence databases. The principle steps involved in the design of probes are: the alignment of rRNA gene sequences; the identification of sequence idiosyncrasies; the synthesis and labelling of complementary nucleic acid probes; and the experimental evaluation and optimization of the probe specificities and assay sensitivities using cultured reference strains.

Nucleic acid hybridization can be performed in a variety of formats. The procedure typically entails the extraction of whole-cell DNA or RNA from the sample that is subsequently fixed to a nylon or nitrocellulose membrane. Alternatively, bacterial colonies can be replica-plated from agar plates to membranes and their nucleic acids exposed following lysis for subsequent hybridization. Whereas the former direct approach is usually inadequate due to high detection limits and large sample volumes that are impractical for most hybridization protocols without further pathogen concentration, the latter approach is time-consuming and cannot be applied to nonculturable organisms. Consequently, the use of whole-cell fluorescent in situ hybridization (FISH) as a method for the direct detection of bacteria in water samples has increased in popularity. The method typically combines membrane filtration, resuscitation of the bacteria on membranes and hybridization with rRNA-targeted probes. During FISH, the morphology of the cells in the sample has to be stabilized in order to maintain the morphological integrity of the cells under harsh hybridization conditions. The cell walls and membranes have to be permeabilized to allow free penetration of fluorescent oligonucleotides to the intracellular rRNA. This can be achieved with fixatives such as aldehydes and/or alcohols. The membranes are hybridized in hybridization solution containing a fluorescently-labelled oligonucleotide. Following incubation at the hybridization temperature for one to several hours, to allow the probe to bind to complementary rRNA sequences, washing steps are used to remove unbound or the non-specifically bound fluorescent probe, and the hybridized cells are then detected by epifluorescence microscopy. Probes will only bind correctly under defined hybridization conditions and the optimization of hybridization and washing conditions is as important as the probe design. Several probes labelled with spectrally different fluorochromes can be simultaneously used on one sample, while counterstaining of the fixed cells with DAPI (4',6-diamidino-2-phenylindole) allows total counts to be made. Alternatively, depending on the concentration of targeted cells in the sample and to increase resolution, FISH hybridization can be performed in suspension and detection can be performed by means of flow cytometry, which enables quantification of the fluorescence intensities for each target-probe hybrid.

Although FISH is currently considered a highly specific cellular detection method and is relatively easy to perform, the procedure may have some limitations when applied to the detection of nutrient-starved bacterial cells disseminated in drinking waters. Generally, probes give a strong signal only if cells are metabolically active and contain large numbers of ribosomes and target rRNA. Conversely, low cellular ribosome contents may result in weak fluorescent hybridization signals. Another area of concern relates to the viability of the detected cells. Although it has been reported that the rRNA content of bacterial cells can be directly correlated with the growth rate, some reports have indicated that a small number of rRNA molecules can remain for a relatively long period after the loss of culturability, thus resulting in false positives. One possible way to overcome this issue is to couple direct viable count techniques with FISH detection.

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