from Theron et al. in Nanotechnology in Water Treatment Applications

As a consequence of the speed, specificity and low cost of the PCR, the procedure has become one of the most widely used assays for direct detection of low levels of pathogenic microbes in environmental samples. The PCR assay can be used to selectively amplify, to detectable levels, nucleic acid sequences associated with pathogens that might be present in low numbers in water samples. PCR is a process in which target DNA, synthetic oligonucleotide primers, a thermostable DNA polymerase and the DNA subunits are combined in a microcentrifuge tube and subjected to the temperature changes needed for the DNA duplication to occur. During the PCR process, different temperatures are used to facilitate DNA denaturation, annealing of the oligonucleotide primers to the target DNA and extension of the primers across the target sequence. These cycles are repeated many times, thus resulting in increasingly greater quantities of target sequence. Under ideal conditions, the PCR can generate millions of copies of a single DNA molecule in just 20 to 30 repetitions of the temperature cycle, with each cycle requiring only a few minutes. The PCR-amplified products can be detected by means of techniques such as electrophoresis on agarose gels and after staining of the amplification products by a fluorochrome dye or by hybridization with a labelled probe.

There are several essential steps in the development and application of PCR for successful detection of pathogens in water samples. The key steps include: identification and selection of oligonucleotide primers for target genomic sequences; testing of selected primers for sensitivity, specificity and selectivity; purification and concentration of the pathogens in environmental sample concentrates to enable efficient and reliable enzymatic amplification of low numbers of target genomic sequences; and testing of the methods for their applicability to natural pathogenic strains and actual field samples. By using PCR, a selected gene sequence specific to a group of organisms or a single species can be selectively amplified. Various primers have thus been described to amplify fragments of rRNA operons in order to detect specific organisms or groups of organisms in environmental samples. As in the case of designing oligonucleotide probes for hybridization purposes, selection of oligonucleotide primers for target pathogens requires that sequence data be available. Of particular importance is the type and function of the nucleic acid target; its length and location; and the extent to which its sequence is related to that of other, nontarget but genetically related microbes. It is essential to select oligonucleotides having an appropriate length (20 to 30 nucleotides); desired sequence composition for specificity and selectivity; and appropriate melting and annealing temperatures to prevent the formation of undesirable secondary structures, primer-dimers and other artifacts that would interfere with successful PCR.

There exist many variations of the basic PCR technique. The sensitivity and specificity of the PCR may be improved by adopting a nested approach. Nested PCR involves two consecutive rounds of PCR amplification. The first round of amplification is performed with group- or organism-specific primers, whilst the second round of amplification uses the initially amplified product as the template for another round of annealing and extension with different primers. The use of nested primers provides an additional level of specificity since the second round of PCR amplification can only be performed if the correct sequence (complementary to the nested primers) was amplified during the first round. Alternatively, nucleic acid hybridization can be performed using highly specific oligoprobes that would hybridize only with amplicons from a single pathogen type or strain. However, nested PCR permits a more rapid detection (6 to 8 h) compared to confirmation of the correct sequence amplification by probe hybridization (few days). Another variation of the PCR technique, namely multiplex PCR, allows the simultaneous detection of more than one target organism in a single PCR using multiple pairs of primers designed to be specific for different target organisms. However, multiplex PCR may not perform well with all primer blends as the composition and length of primer oligonucleotide, as well as the size of the amplified fragments, may influence each PCR amplification. Since the PCR method cannot be used directly for the amplification of an RNA target sequence, a complementary DNA copy (cDNA) thereof must first be synthesized. This reaction is catalyzed by the enzyme, reverse transcriptase (RT), which is able to synthesize DNA from the RNA template in the presence of specific primers and DNA subunits. The single-stranded cDNA is a suitable target for PCR amplification by making use of the same or a different set of primers. RT-PCR has emerged as a sensitive and specific approach for the detection of enteric viruses containing RNA genomes.

Although the basic PCR method is both specific and sensitive, such standard PCR reactions are not quantitative. To obtain quantitative data from PCR-based analyses, statistical methods based on most probable number (MPN) estimations have been used. In MPN-PCR, DNA extracts are diluted before PCR amplification and limits are set on the number of genes in the sample by reference to known control dilutions. Another way to quantify PCR-amplified products for comparison is to include an internal control in the PCR reaction. Here, a known amount of target DNA is added to a PCR reaction containing DNA from the mixed microbial population. The known target DNA is complementary to the same primers and thus competes with the target sequences in the PCR reaction mixture. By preparing a dilution series of the known and unknown DNA species, it is possible to quantify the amount of product produced from the complementary gene in the extracted DNA. The known DNA target can be generated by cloning the gene of interest or purifying the PCR-amplified product after which a deletion is introduced to give a differently sized PCR product.

An alternative PCR assay for the direct enumeration of targeted cells was reported by Tani et al., who modified the standard PCR protocol so that nucleic acid sequences can be amplified in situ. This new PCR method was successfully applied to the direct enumeration of E. coli from a freshwater sample. With proper fixation and permeabilization conditions, the oligonucleotide primers and other reaction components are able to diffuse into the cells, and, upon thermal cycling, amplify a specific target sequence present in the cell. PCR products were labelled by digoxigenin during the amplification process and anti-digoxigenin antibodies conjugated with fluorescent dye were used for detection by epifluorescence microscopy. This approach allows direct visualization of the fluorescent amplification products at a single-cell level and consequently, direct enumeration of cells. Even though in situ PCR seems promising, it has not been used for routine detection and enumeration of microorganisms in water, as the results showed a weak fluorescence intensity signal of targeted cells.

Another promising approach for quantifying the number of cells is real-time quantitative PCR, which consists of monitoring fluorescently-labelled PCR products as they are being amplified. The fluorescent signal can be generated by using an intercalating fluorescent dye (e.g., SYBR Green I or SYBR Gold) or a probe system (e.g., TaqMan). The use of intercalating dyes is the simplest and least costly approach and involves adding the fluorescent dye directly to the PCR. These dyes undergo a conformational change to become a more efficient fluorophore on binding to double stranded DNA (dsDNA). Although SYBR Green I-based assays are very sensitive, the primer's specificity for the target is crucial as any dsDNA is detected, including any primer artifacts, which can lead to false positive results. Moreover, multiplex reactions are impractical since the dye binds to all dsDNA. The TaqMan approach depends on oligonucleotide probes complementary to a sequence located between the two primers used for PCR amplification. At one end of the probe a fluorescent reporter dye is conjugated, whilst at the other terminus there is a quencher that may be another fluorophore (also called dark-quencher). In effect the structure possesses two dyes in close proximity and in this configuration the fluorescence of one (the reporter) is quenched by the other through FRET (fluorescence resonance energy transfer). During the extension step of PCR the DNA polymerase degrades the bound TaqMan probe, using its inherent 5'-3' exonucleolytic activity, and thus results in the separation of reporter from quencher and an increase in fluorescence emission of the reporter molecule. Although this approach is less prone to false positive results compared to the use of intercalating fluorescent dyes, it is more expensive due to the requirement of the probe molecule. However, by choosing the fluorophores astutely it is possible to perform multiplex PCR. During real-time PCR, irrespective of the approach used, the accumulation of amplified product is measured automatically at each PCR cycle. The amount of target sequence is deduced from the number of PCR cycles (threshold cycle or Ct) required to cross a fixed point above a baseline, using a standard curve as reference. External quantification standards for the construction of standard curves of Ct versus copy number usually consist of the target sequence cloned into a plasmid or DNA extracted from cultured cells where the concentration or copy number of the target can be determined accurately.

Although there are numerous advantages associated with PCR as detection tool, standard PCR cannot, however, be used to detect the infectious state of an organism - only the presence or absence of pathogen-specific DNA or RNA. Yet, this viability concept is fundamental for interpreting the result in terms of public health when dealing with water samples. The PCR technique must consequently be associated with a viability test. To overcome this limitation, an indirect approach has been developed for assessing the viability of PCR-detected bacteria from water samples. This method is based on the analysis of each sample before and after a culture step in a nonselective medium: an increase in the PCR-amplified product after cultivation indicates the occurrence of bacterial multiplication and thus demonstrates the viability of the detected bacteria. Recently, a PCR-based approach to limit detection to intact (viable) cells with an active metabolism has been reported. The approach is based on the use of ethidium monoazide (EMA), which is suggested to enter only membrane-compromised cells (considered "dead"). Once inside membrane-compromised cells, EMA intercalates into the DNA and is covalently bound to the DNA after exposure of the treated samples to bright visible light, whilst the unbound EMA, which remains free in solution, is simultaneously inactivated by reacting with water molecules. The EMA treatment is followed by extraction of genomic DNA and analysis by PCR. The result of treatment is that only unmodified DNA from intact cells whose DNA was not cross-linked with EMA can be amplified, whereas PCR amplification of modified DNA from membrane-compromised cells is efficiently suppressed. Treatment was thus suggested to lead to the exclusion of cells with damaged membranes from analysis.

Despite its advantages, accurate characterization or identification of microbes by PCR is influenced by the same bias and variations that are inherent in many nucleic acid techniques. The main concerns are biased nucleic acid extraction (e.g., efficiency of extraction or cell lysis if using whole-cell methods), degradation of nucleic acids by nucleases and primer reactivity (i.e. sensitivity, specificity and accessibility). Additionally, a frequently encountered limitation inherent to PCR analysis of environmental samples is the inhibition of the enzymatic reaction. Whereas humic substances are known to inhibit the DNA polymerase enzyme, colloidal matter has a high affinity for DNA. The presence of these elements in a water sample can therefore considerably decrease the amplification yield of PCR applied to the detection of greatly diluted bacteria. Consequently, for PCR or RT-PCR, the extracted target nucleic acid is purified by protocols utilizing, for example, Sephadex, Chelex or CTAB.

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