"reviews and illustrates the use of quantitative real-time PCR for a number of different purposes. It covers the basic process as well as the technology that has improved its performance, while also exploring the various scientific fields that use this technique routinely. It provides a complete description of what scientists need to design and perform a quantitative PCR ... useful to scientists in many different types of laboratories, including public health, environmental, clinical diagnostic, and food industry. It also can be useful to students and young investigators as well as experienced scientists. The authors clearly are familiar with the development and application of quantitative PCR and share their experience here ... This useful book is filled with valuable information for any laboratory using PCR to detect microbial agents and will serve as a resource for many years to come. " from Rebecca T. Horvat (University of Kansas, USA) writing in Doodys read more ...

Quantitative Real-time PCR in Applied Microbiology Edited by: Martin Filion ISBN: 978-1-908230-01-0 Publisher: Caister Academic Press Publication Date: May 2012 Cover: hardback "useful book ... filled with valuable information" (Doodys)

from Theron et al. in Nanotechnology in Water Treatment Applications

In contrast to gold nanoparticles and QDs, magnetic nanoparticles have not been used in many biological applications. Nevertheless, advances in the synthesis of monodispersed magnetic nanoparticles, ranging in size from 2 to 20 nm, has provided a basis from which to explore applications of magnetic nanoparticles in diagnostics. Magnetic nanoparticles are produced from materials that can be strongly attracted by magnets or be magnetized. They can be prepared in the form of single domain or superparamagnetic (Fe₃O₄), greigite (Fe₃S₄), maghemite (gamma-Fe₂O₃), and various types of ferrites (MeO.Fe₂O₃, where Me = Ni, Co, Mg, Zn, Mn, etc.). Bound to biorecognition molecules, magnetic nanoparticles can be used to facilitate the separation, purification and concentration of different biomolecules. To do so, biorecognition molecules such as antibodies can be immobilized on the surface of magnetic nanoparticles through covalent or electrostatic interactions. After reacting these magnetic nanoparticles with sample solutions, targeted molecules can be bound by or captured on the surface of these magnetic nanoparticles. By applying a magnetic field, these nanoparticles can subsequently be concentrated and separated from the bulk solution and identified.

Biofunctional magnetic nanoparticles, in which thiolated vancomycin was attached to FePt nanoparticles, have been used to capture and detect of a wide range of bacteria at very low concentrations within 60 min. These included capturing and detection of Staphylococcus aureus at 8 cfu/ml, S. epidermidis at 10 cfu/ml, Enterococcus faecalis at 26 cfu/ml, and E. coli at 15 cfu/ml. Although the sensitivity achieved using magnetic nanoparticles is comparable to PCR-based assays, the direct capture protocol is faster than PCR when the bacterium count is low since it obviates the need for pre-enrichment of bacteria through culturing. In an alternative approach, Ho et al. reported combining biofunctional magnetic nanoparticles with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to detect pathogenic bacteria in water. Biofunctional nanoparticles were fabricated by attaching human immunoglobulin (IgG), which binds selectively to IgG-binding sites on the cell walls of pathogens, onto the surfaces of magnetite (Fe₃O₄) nanoparticles. Using this assay, both S. saprophyticus and S. aureus were detected at concentrations of 3 x 10⁵ cfu/ml in aqueous sample solutions. Measuring adenosine triphosphate (ATP) bioluminescence of bacteria captured onto magnetic nanoparticles is another proposed method for detecting microorganisms. E coli was detected in milk by Cheng et al. within a short period (1 h) and with a low detection limit (20 cfu/ml).

Biofunctional magnetic glyconanoparticles have also been engineered by covently binding unmodified monosaccharide d-mannose onto iron oxide nanoparticles. These particles had the ability to recognize mannose-specific receptor sites on E. coli. Magnetic nanoparticles have been developed to sequester DNA in water and capture the DNA-nanoparticles complexes by the application of high-gradient magnetic separation. Modifying magnetite clusters with poly(hexamethylene biguanide)- and polyethyleneimine resulted in strong cationic nanoparticles which enabled the binding with DNA molecules through electrostatic forces. The cationic nanoparticles can also serve as a disinfectant by binding to the negatively charged cell envelopes of bacteria. These particles were colloidally stable in fresh and ocean water for weeks at a pH <= 10.

Magnetic microparticle-antibody conjugates (Dynabeads) are commercially available and kits have been developed for the detection of Legionella species, Cryptosporidium oocysts and Giardia cysts from concentrated water samples. Dynabeads are also available for the detection of E. coli, Salmonella and Listeria species; however the samples must be grown for 6 - 8 h in a pre-enrichment broth. Streptavidin coated Dynabeads allow researchers to design their own magnetic microparticle-antibody conjugates for specialized assays (www.invitrogen.com). Biotinylated organism-specific antibodies will bind covalently onto the streptavidin coated Dynabeads. A wide range of biotin-labeled antibodies are available from companies such as Abcam (www.abcam.com).

Despite the promise shown by biofunctional magnetic nanoparticles, some challenges regarding their widespread use have yet to be overcome. In addition to requiring a robust surface chemistry to attach bioactive molecules onto magnetic nanoparticles without laborious synthetic efforts, more precise control of the numbers and orientations of the molecules on the surfaces of magnetic nanoparticles is also required.

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.

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.

from Theron et al. in Nanotechnology in Water Treatment Applications

Significant advances in the detection of sequence-specific nucleic acid hybridization have been achieved using microarrays. Microarrays are glass microslides or nylon membranes containing a high density of immobilized nucleic acids (genomic DNA, cDNA or oligonucleotides) in an ordered two-dimensional matrix. Microarrays can be prepared by synthesizing DNA in situ on a glass surface using combinational chemistry or by robotic microdeposition of cDNAs (0.5- to 2-kb) amplified by PCR. The sample DNA, usually bound to a fluorescent or enzyme label, is exposed to the microarray and hybridizes with the target sequences. The detection of the probe-target hybrid at each spot on the array is achieved either by direct fluorescence scanning or enzyme-mediated detection yielding a semi-quantitative result. Advantages of DNA microarray technology, as compared to other techniques, include the small size of the array allowing for a higher sensitivity, the ability to simultaneously detect diverse individual sequences in complex DNA samples, and the capacity to do comparative analysis of a large number of samples. Indeed, PCR-microarrays have been used to measure relative concentrations of microbes from water, to characterize microbial communities from environmental samples and to detect bacterial pathogens from a variety of sources, including water. However, obstacles such as sample size, matrix-associated inhibitors, nonspecific binding and cross-hybridization must be overcome before microarrays can be used generally for the detection and differentiation of pathogens in environmental samples. Moreover, microarrays cannot distinguish between nonviable, culturable and viable but nonculturable (VBNC) cells since DNA can persist for long periods of time after the death of cells. The ability to discriminate between living and nonliving cells is important in order to interpret the risk associated with the detection of pathogenic microbes (especially in environmental samples). The use of mRNA, which is highly labile with a short half-life, or the use of highly expressed targets may be selected to provide sensitive analyses.