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Australasian Biotechnology (backfiles)
AusBiotech
ISSN: 1036-7128
Vol. 6, Num. 5, 1996
Australasian Biotechnology,
Volume 6 Number 5, September/October 1996, pp.285-294

Nucleic Acid Sequence Detection Systems: Revolutionary Automation for Monitoring and Reporting PCR Products

Brant J Bassam^1, Traci Allen^2, Susan Flood^2, Junko Stevens^2, Paul Wyatt^2, and Kenneth J Livak^2

Perkin-Elmer, Applied Biosystems Division:
^1 1270 Ferntree Gully Rd., Scoresby, Victoria 3179, Australia;
^2 850 Lincoln Centre Dr., Foster City, California 94404, U.S.A.


Code Number: AU96015
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    Text: 37.0K
    Graphics: Photographs (jpg) - 431.6K

[ALL TABLES AND FIGURES AT END OF TEXT]

Abstract

Recent technological advances at PE-Applied Biosystems have made fully-automated real-time detection of specific PCR products possible. Both the 7700 and 7200 sequence detection systems integrate four major elements: (1) fluorogenic chemistry for target-specific oligonucleotide probes; (2) an exploitation of the polymerisation-dependant 5' nuclease activity of the DNA polymerase; (3) instrumentation to measure fluorescence signal within a closed PCR reaction vessel; (4) software that processes and analyses the data. Dedicated systems are available for simple end-point analysis of accumulated PCR product and real-time analysis to accurately determine initial target copy number. Real-time analysis also allows the reaction kinetics of a PCR to be interrogated and provides useful optimisation data. Multiple fluorophores are used for signal normalisation, PCR controls, and multiplex applications. These systems have been designed for cost-effectiveness and high-throughput, and should revolutionise nucleic acid sequence detection and genetic screening studies.

Introduction

There is no doubt that the sensitivity, specificity, and simplicity of the PCR has had enormous impact on modern molecular biology. Despite this, PCR-based methods are not widely used for high-throughput, routine testing. This is primarily because existing assays are labour-intensive, relatively time-consuming, and expensive compared to traditional methods. Also, the amplification process itself remains somewhat enigmatic. Consequently, there is a level of distrust for the "black box" nature of the PCR leading to often unreliable and improperly-optimised assays with poor experimental controls.

The significant processing normally required to detect PCR products and analyse results poses significant risks for "carryover" contamination. Carryover produces false-positive results when amplified products from previous experiments spread throughout a laboratory. False-negative results can also be problematic and can arise from pipetting errors and poorly-prepared sample material. This can be particularly troublesome with certain "dirty" sample types in which inhibitors copurify with the nucleic acid sample and stifle the PCR.

The ideal system for high-throughput testing would involve a truly "homogeneous" PCR assay, ie. one in which the dual processes of amplification and detection are done seamlessly and concurrently. This would allow specific PCR products to be monitored within a closed reaction vessel and total post-PCR automation to be achieved.

Higuchi et al (1992; 1993) developed the first homogeneous real-time method for detecting PCR products. They exploited the fact that fluorescence of the intercalator dye ethidium bromide increases in the presence of double-stranded DNA (dsDNA). By monitoring increasing fluorescence as dsDNA product accumulated during amplification, they could continuously follow the progress and kinetics of a PCR reaction. Despite the obvious advantages of this intercalator dye system, its major drawback is its non-specificity; it cannot discriminate between the PCR product of interest and the common PCR "noise" from mis-priming and primer-dimer artefacts.

An alternative probe-based strategy for detecting a specific PCR product was pioneered by Holland et al in 1991. They exploited the 5'-3' nucleolytic activity of Taq DNA polymerase and demonstrated that it could act on a probe specifically targeted to a PCR product during the amplification reaction. To accomplish this, they first designed a reporter oligonucleotide that would hybridise to a target PCR product. After chemically blocking the 3' end to prevent it acting as a primer and labelling the 5' end with ^32P, this reporter oligo was simply included with the normal PCR reagents. The reporter oligo annealed to the target sequence of the accumulating PCR product, forming a structure that could be cleaved by the 5' nuclease activity of the polymerase as it extended the upstream PCR primer into the vicinity of the annealed oligo. The cleaved remains of the oligo were later separated and detected using thin layer chromatography. The dependence on hybridisation and polymerisation for oligo cleavage ensured that signal was generated only if the intended amplification had occurred; unhybridised probe is unaffected by the enzyme. Thus the usefulness of this system is its ability to report the presence of a particular PCR product while avoiding background PCR noise. Nevertheless, a major drawback was the significant post-PCR handling still required.

The 5' nuclease assay of Holland et al (1991) was enhanced by Lee et al (1993) into a homogeneous system for PCR amplification and specific product detection. They developed an improved reporter oligonucleotide design incorporating fluorescent dyes for signal detection. This made it possible to eliminate post-PCR processing, as Higuchi et al (1992; 1993) had done, but with the added advantage that only specific amplification products were detected. It is continued development of the 5' nuclease assay (Holland et al 1991; Gelfand et al 1993; Lee 1993; Livak et al 1995; Livak 1996) that forms the basis of the sequence detection systems described here.

The fluorogenic reporter probe: the heart of the system

The fluorogenic 5' nuclease assay uses only a single fluorescent oligonucleotide probe in addition to standard PCR reaction components and controls. A great deal of work has already been done on the design and chemistry of these probes, an appreciation of which helps enormously in understanding the assay.

Current probe design makes use of an energy transfer effect between matched fluorescent dyes. This principle, known as F rster resonance energy transfer through space or FRET (F rster, 1948; Lakowicz, 1983), has been widely used as a biological research tool (Stryer and Haugland, 1967; Stryer, 1978). With FRET, light energy adsorbed by a high-energy chromophore is transferred to a nearby chromophore of lower energy subject to certain geometric and spectroscopic constraints, the most important of which is the distance between the two chromophores.

For FRET to work as a reporter system in the PCR, probes are labelled with two fluorescent dyes. One dye acts as a "reporter" and one as a "quencher": the reporter transfers fluorescent energy to the quencher by FRET when they are in close proximity. Signal is generated when the hybridised oligo probe is cleaved by the DNA polymerase and the dyes are separated. This separation negates FRET effects and fluorescent signal emitted by the reporter dye measurably increases (Fig. 1).

In intact probes, close proximity of the quencher and reporter dyes is critical for FRET. Initial probe designs covalently linked the reporter dye to the 5' end of the oligo and the quencher to a linker-arm-modified nucleotide also close to the 5' end to ensure proximity of the dyes. However, it is now standard to place the quencher at the distal 3' end of the probe (Livak et al, 1995). This may seem contradictory to FRET requirements since doing so seems to maximise rather than minimise the distance between the dyes. Nevertheless, the dyes do approach each other closely since the single stranded oligo is flexible enough to bend and contort in solution. These contortions occur quickly compared to the lifetime of the excited state of the reporter, and the quenching observed is a time-resolved average of a population of probes in all bending configurations. There is no doubt that having dyes at opposite ends of the probe can markedly reduce net quenching and therefore overall signal strength. On the other hand, placing both fluorophores close together at the 5' end of the probe can result in probe cleavage downstream from both dyes resulting in no signal whatsoever. Thus, the loss of net quenching due to terminal placement of the dyes is more than offset by the fact that any cleavage event will generate signal.

The mechanism of cleavage warrants some discussion. Probes are not digested nucleotide-by-nucleotide. Rather, the polymerase acts by first destabilising the 5' end of the hybridised probe to form a fork-like structure before cleaving the oligo a variable number of nucleotides into the probe. This is why the mechanism is properly referred to as "the forklike-structure-dependent, polymerisation-associated, 5' to 3' nuclease activity of DNA polymerase." Thus, probes are actually cleaved into short chunks. This quickly destabilises the portion of the probe still hybridised so that it dissociates back into solution. Strand extension can then be completed with no inhibition of the overall PCR process.

Several dyes are currently used in the fluorogenic 5' nuclease assay. FAM (6-carboxyfluorescein), TET (tetrachloro-6- carboxyfluorescein), and JOE (2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein) can act as reporter dyes, each of which uses TAMRA (6-carboxy-tetramethlyrhodamine) as a quencher. A modified ROX (6-carboxy-X-rhodamine) dye is also included in the PCR reaction buffer as a passive internal reference for signal normalisation.

Three main factors affect the performance of probes: the efficiency of probe hybridisation; the efficiency of reporter dye quenching; and the efficiency of probe cleavage by the polymerase. For efficient hybridisation, the fluorogenic probe needs to have a melting temperature (Tm) higher than that of the PCR primers used. This is because primer-template hybrids are quickly stabilised as they are extended by the DNA polymerase; whereas, the fluorogenic probe is not extended and therefore not stabilised. The probe is purposely blocked at its 3' terminus so that it will not act as a primer. The higher Tm of the probes thus compensates for their relative instability. The recommended Tm of the probe is 70^oC and compatible PCR primers should have a Tm approximately 10^oC lower. Probe length is normally optimal at between 20-25 nucleotides (nt). Besides having a reduced synthesis efficiency, probes longer than about 30 nt risk forming inter- or intra- molecular structures that interfere with probe synthesis, flexibility, hybridisation, cleavage, as well as DNA amplification.

Just as there are important guidelines for PCR primer design, there are similar considerations for probe design. For example, the G/C ratio of a probe sequence can affect hybridisation efficiency. For unknown reasons, probes with a higher proportion of C bases over G bases usually perform better. While the G + C content of complementary strands is identical, one strand often has more C bases and the other more G bases. Since probes can be designed for either strand of the PCR product, it is better to use the strand with more C bases as the probe. Other guidelines include avoiding runs of identical nucleotides (especially four or more Gs), avoiding complementarity or overlap between the probe and primers, and avoiding a G at the 5' end of the probe.

Dedicated software for probe and primer design is now available (Primer Express^TM, P-E Applied Biosystems) to simplify this process. Because probes have such specific requirements, it makes practical and economic sense to design the probe first and the PCR primers afterwards: it is cheaper and simpler to experiment with several sets of primers, rather than several fluorescent probes when optimising an assay. Also, our experience indicates that finding a more efficient pair of primers has a much bigger positive effect on overall fluorescent signal than changing probe sequences.

Considerations for the PCR

Containment and contamination issues cannot be overstated for the PCR. Even though amplification and detection are done within a sealed tube, standard reaction components for the fluorogenic 5' nuclease assay integrate the uracil N-glycosylase (UNG) carryover prevention strategy. In this strategy, dUTP is substituted for TTP during DNA polymerisation. This allows PCR products from previous experiments to be differentiated from fresh template DNA in later experiments. UNG recognises and enzymatically degrades the uracil-containing products before they can act as carryover templates in later experiments. UNG is not active at 55^oC or above, so annealing/extend temperatures of at least 55^oC are used in the fluorogenic 5' nuclease assay.

For optimal PCR fidelity, standard reactions integrate a "hot start" strategy (Erlich et al, 1991). Hot starts overcome problems of nonspecific primer annealing and extension that occur when reaction components are mixed at room temperature. Reliable hot starts are easily achieved by simply using AmpliTaq Gold^TM DNA polymerase (Birch et al 1996). AmpliTaq Gold DNA polymerase is the first of a new generation of heat-activated enzymes. It is a modified form of the original AmpliTaq DNA polymerase that is activated only after exposure to elevated temperature. The polymerase is thus prevented from acting on reaction components until thermocycling begins. This effectively creates hot start conditions which are "transparent" to the user.

Regarding sensitivity, it is important to differentiate between the starting copy number of the target sequence and the amount of PCR product required for accurate measurement. Determining the starting copy number is primarily a function of the PCR; whereas, the amount of product required for measurement is a function of the detection system. To achieve adequate signal you simply perform as many PCR cycles as necessary to generate sufficient hydrolysed probe. Very low copy number detection/quantitation is not uncommon but may require optimisation. As a rule, if the product of interest can be visualised in an ethidium bromide stained agarose gel, then it is amenable to the fluorogenic 5' nuclease assay.

Instrumentation

First generation instrumentation for the fluorogenic 5' nuclease assay consisted of a modified version of the model LS-50B luminescence spectrometer and custom PC-based software. It is still the instrument of choice when the flexibility to do a wide variety of other fluorescent or luminescent studies is an important consideration. The TaqMan^TM LS-50B PCR Detection System can read from either open microtitre plates or closed PCR-reaction tubes. Second-generation instruments dedicated to the fluorogenic 5' nuclease assay are now available: the ABI PRISM 7700 and 7200 Sequence Detectors (Fig. 2).

The ABI PRISM^TM 7700 Sequence Detection System was designed for "real time" monitoring of the fluorogenic 5' nuclease assay by measuring fluorescent signal as it is generated during thermal cycling. It integrates a thermal cycler; a laser light source; a fibre-optic network to distribute the laser light to, and the fluorescence signal from, each reaction tube; a cooled CCD camera detector; and a Macintosh computer with dedicated software. In less than 7 seconds, the system collects fluorescent emission over 32 discrete spectral bands between 500 nm and 660 nm from all 96 reaction tubes sealed inside the thermocycler block. After collecting the signals, the software first calculates the contribution of each component dye to the spectrum and normalises the reporter signal against the standard ROX reference dye. Peak normalised signals are averaged and plotted versus cycle number to produce "amplification plots" of PCR products. Any part of the amplification process can be monitored providing an "information supermarket" of PCR data. The 7700 can be used for quantitative PCR, very high throughput end-point detection, and PCR kinetics and optimisation studies.

The ABI PRISM^TM 7200 Sequence Detection System was designed for "end point" detection: it measures fluorescent signal after thermal cycling has been completed. Like the 7700 system, detection is done from within sealed reaction tubes. The only post-PCR processing required is simply to transfer the reaction tubes from the 96-well thermal cycler to the automated sample drawer of the instrument. The 7200 takes about 15 minutes to measure the entire 32-band spectrum from each tube between 500 nm and 660 nm using a xenon flash lamp light source. Data is also processed on a Macintosh computer. The 7200 is designed for routine pathogen detection and allelic discrimination assays.

Pathogen Detection

The detection of microbial pathogens in food, environmental, and other samples is a critical, time sensitive function for many laboratories. Turnaround time is especially important to the food industry, for example. Faster tests mean storage cost savings and longer product selling times. Traditionally, the method of choice for pathogen detection has been culture-based. Existing culture tests take between 2 and 7 days to complete. Popular immunoassays and biochemical tests (including latex agglutination, immunodiffusion/antibody capture, ELISA/EIA, and dipstick assays) take 2 to 4 days to complete. The PCR-based sequence detection systems described here are ideal for routine pathogen detection and reduce total assay times to less than 24 hours.

Both the 7700 and 7200 instrument systems can be used for high throughput pathogen detection. They offer a level of reliability and productivity not possible with other detection methods. All the reagents and protocols needed to perform research assays are included with the TaqMan^TM PCR Reagent Kit. In addition, a wide range of kits will be available for detecting specific microorganisms, including E. coli strains, Salmonella, Listeria, Giardia, and Cryptosporidium from food, dairy and other environmental samples. The fluorogenic 5' nuclease assay thus provides a common platform for a wide range of tests.

The first kit available for testing food and environmental samples is the TaqMan^TM Salmonella PCR Amplification/Detection Kit. It has the PCR reagents supplied as a master mix and includes an internal amplification control to enable discrimination between true negatives and false negatives. It includes protocols for a 16 hour pre-enrichment step and a sample preparation step (0.5 to 2 hrs), reagents for PCR and detection (2.5 hrs), and dedicated software for data analysis (5 min). The data analysis provides answers in a simple Yes/No/Retest format (Table 1). When there is a significant increase in signal from the Salmonella probe, the software reports "Yes." When there is no significant increase in Salmonella signal, but there is positive signal from the internal amplification control, this is a true negative and the software reports "No." The software reports "Retest" when there is no significant increase in either Salmonella or internal control signal.

Allelic Discrimination

Allelic discrimination has traditionally been done using labour-intensive procedures such as enzymatic restriction analysis of PCR products, single strand conformational polymorphism (SSCP) analyses, competitive oligonucleotide priming (COP) assays, or by direct DNA sequencing. It is now possible to routinely differentiate between two forms of a gene differing by as little as a single base using the fluorogenic 5' nuclease assay. Two fluorescently-labelled probes specific for each allele are added together to a reaction. The probe for the first allele is normally labelled with FAM and the probe for the second allele with TET. Discrimination between each allele sequence is driven primarily by competition between homologous and heterologous probe hybridisation events; the homologous event hybridising better. An increase in the FAM signal indicates the sample was homozygous for the first allele, TET signal indicates homozygosity for the second. A mixture of both FAM and TET signals characterises an individual heterozygous for both alleles. Lee et al (1993) distinguished between deltaF508 and normal alleles of the human cystic fibrosis gene using this approach. Livak et al (1995) used a more recent probe design to similarly distinguish the single base -23 A/T diallelic polymorphism of the human insulin gene associated with type 1 diabetes. Dedicated software for allelic discrimination is used to normalise data for well-to-well variation and background fluorescence. As shown in Fig. 3, this makes allelic discrimination data output clear and highly reproducible.

Strand-specific mutation analysis, or haplotyping, is useful for risk-assessment of genetic abnormality carrier status. Existing methods use allele-specific primers and involve time-consuming agarose gel electrophoresis. The fluorogenic 5' nuclease assay provides a way to streamline haplotyping. By using allele-specific primers to assess one polymorphic site and fluorogenic probes to assess a second polymorphic site on the same amplicon, haplotypes can be determined without lengthy post-PCR processing.

Quantitative PCR

There are many applications in molecular biology and biomedical research for measuring the concentration of particular DNA or RNA sequences in native samples. In some applications, such as gene expression studies, determining absolute amounts may not be required. Instead, sensitive and quantitative measurements of relative concentration changes are required.

The ability of the 7700 system to monitor the progress of a PCR reaction in real-time has completely revolutionised PCR-based nucleic acid quantitation. Traditional methods examine the final amount of PCR product accumulated after a fixed number of cycles. Real-time monitoring can define the point during cycling at which amplification of a PCR product is first detectable. This has important ramifications on precision and reproducibility.

Early in the PCR, reagents are not limiting and amplification is uniform and exponential. As cycling progresses however, target amplification slows until a plateau is reached and there is little or no net increase in PCR product. The sensitive fluorescence detection of the 7700 allows products to be detected very early in the exponential phase, usually well before any one reagent becomes limiting. The parameter C[T] (threshold cycle) is defined as the fractional cycle number at which the reporter fluorescence generated by cleavage of the probe passes a fixed threshold level above baseline. As illustrated in Fig. 4a, the higher the starting copy number of nucleic acid target, the fewer the amplification cycles needed before a significant increase in fluorescence is observed and the lower the C[T] value. Measuring the starting copy number in an unknown sample is simply done by determining the C[T] and reading the corresponding starting copy number of the target from a standard curve of C[T] vs. copy number such as that shown in Fig. 4b.

C[T] values allow for such precise and reproducible quantitation measurements because they are obtained at a stage in the reaction where reagents are not limiting. The amount of PCR product observed at the end of a reaction, on the other hand, is highly sensitive to even tiny variations in reaction components. Endpoint measurements are usually made when the reaction has passed the exponential phase and a slight variation in a limiting component can drastically effect the final amount of a product. This is because side reactions, such as the formation of primer dimers, can consume reagents to different extents from tube to tube. Indeed, samples with higher starting copy number may end up with less accumulated product than samples with lower starting copy number. This is graphically demonstrated in Fig. 5, which shows how the overall change in reporter signal for 96 replicate PCRs varied widely between cycles 30 and 40, but was very reproducible between cycles 22 and 25, where the C[T] values are determined. C[T] values are also less sensitive than endpoint values to the effects of PCR inhibitors, again because measurements are taken early in the exponential phase where components are not limiting.

The entire process of calculating C[T]s, preparing a standard curve, and determining starting copy number for unknown samples is done by the 7700 software. It is important to realise that data collected beyond the calculated C[T] is immaterial for quantitation purposes; the final amount of accumulated product and the slope of the amplification plot are simply irrelevant. Compared to endpoint measurements, calculating C[T]s greatly expands the dynamic range of quantitation because data is collected at every cycle. Furthermore, the 7700 eliminates post-PCR processing. This increases throughput, reduces the risk of carryover contamination, and removes post-PCR processing as a potential source of error.

Interrogating the PCR

Fluorescent data can be captured from any or all segments of a thermal cycling regime. This extra information can be used to better understand what is happening during PCR amplification. For example, product accumulation during a single cycle can be monitored because multiple fluorescence readings are taken during each extension phase. By noting how reporter fluorescence increases with time, the length of the extension phase can be optimised. An extension phase that is too short causes inefficient amplification and an extension phase that is too long wastes time because the overall duration of the assay is longer than it needs to be.

Current fluorogenic probes position the reporter and quencher dyes at opposite ends of the probe. Because of this, probes exhibit a much higher reporter fluorescence when in the double-stranded hybridised state compared to when they are single stranded and free in solution. This has been suggested as a mechanism for homogeneous detection of nucleic acid hybridisation (Livak et al 1995). Future development of such hybridisation assays could be useful for a variety of applications. Bagwell et al (1994) and later Tyagi and Kramer (1996) describe similar assay systems using more complex probes designed to form single-stranded hairpin-loop structures.

By exploiting the increase in reporter fluorescence observed upon hybridisation, the 7700 can be used to measure the Tm of fluorogenic probes. Existing algorithms for Tm calculation only provide estimations and can be surprisingly inaccurate. By mixing a fluorogenic probe with its complementary strand and using the thermal cycler of the 7700 to provide a slow ramp in temperature, the observed change in reporter fluorescence is actually a melting curve for the probe. This makes it easy to obtain precise measurements of a probe's Tm under actual PCR reaction conditions (salt, Mg, and presence of co-solvents). Knowing a probe's Tm,, the temperature of the anneal/extend phase can be fixed at a lower value, ensuring that the probe will be hybridised as the polymerase extends the PCR primers.

Conclusions

The nucleic acid sequence detection systems described here can automate the entire processes of PCR amplification, product detection, data processing, and results presentation. Using target-specific fluorogenic reporter probes ensures that only amplification of the intended sequence is measured. The ability to detect PCR products through closed tubes greatly reduces the risk of crossover contamination. Total, integrated solutions for pathogen detection, allelic discrimination, quantitation, and optimisation studies are available. Compared to endpoint quantitation methods, real-time PCR offers reproducible results and a large dynamic range. These revolutionary advances provide new and powerful tools for a wide range of PCR-based applications.

    Figure Legends

    Figure 1: Stepwise diagram of fluorogenic 5' nuclease assay. The action of Taq DNA polymerase on the hybridised fluorogenic probe is shown during one extension phase of the PCR.

    Figure 2: The ABI PRISM 7200 (left) and 7700 (right) Sequence Detectors. The 7200 measures fluorescent signal from 96 sealed PCR reaction tubes after cycling is completed. The output is analysed on a Macintosh^TM computer. The 7700 additionally integrates a thermal cycler and laser-driven optics to monitor the course of a PCR in real time.

    Figure 3: Allelic discrimination. Duplicate samples from 43 individuals were typed for a single-base polymorphism (A/C) in the human DNA ligase I gene (LIG1). PCR reactions were performed containing a FAM probe specific for allele A (the A-containing allele) and a TET probe specific for allele B (the C-containing allele). Shown is the output from the allelic detection module of the ABI PRISM^TM Sequence Detection software. This software analyses the end-point fluorescence data from either the 7700 or 7200 and automatically types each sample as an A homozygote, a A/B heterozygote, or B homozygote. Reference reactions were run in wells A1-10 and the experimental samples were run in the remaining wells.

    Figure 4: RT-PCR amplification of RNA over six orders of magnitude. A segment of human GAPDH cDNA was cloned and used as template for in vitro transcription with T7 RNA polymerase. Dilutions of a known amount of this synthetic GAPDH transcript were the templates for RT-PCR using the components of the EZ rTth RNA PCR Kit (Perkin-Elmer). (a) Amplification plots for reactions with starting RNA copy number ranging from 42 to 1.3 x 10^8. Cycle number is plotted versus change in normalised reporter signal (DRn). Four replicates for each copy number were performed, but the data for only one is shown here. (b) Standard curve plotting log starting copy number versus threshold cycle (CT). In this case. The data from all four replicates at each copy number are plotted. (Figure reproduced from Livak, 1996).

    Figure 5: Amplification of a segment of the b-actin gene from human genomic DNA. Samples contained 10 ng human genomic DNA (corresponds to 3300 copies of a single copy gene) and were amplified using the components of the TaqMan^TM PCR Reagent Kit (Perkin-Elmer). (a) Amplification plots of 96 replicates. (b) Detail of cycles 20-28. The abscissa is placed at a DRn value of 0.05 to show the threshold used for calculation of CT. The average final DRn value at cycle 40 is 1.03ñ0.22 (c.v. = 21.4%). The average CT value 24.64ñ0.11. A standard deviation of 0.11 for CT corresponds to a c.v. of 7.9% for calculated starting copy number. (Figure reproduced from Livak, 1996).

--------------------------------------------------------------
Table 1. Typical results obtained using the Salmonella
Taqman^TM assay on a variety of uncontaminated, naturally
contaminated, or artificially contaminated food samples.
--------------------------------------------------------------
Food Sample         Salmonella TaqMan^TM     USDA/FDA Culture 
                    Assay Results (24 hrs)   Methods (5days)
--------------------------------------------------------------
Artificial Egg               Positive           Positive
Scrap Egg                    Positive           Positive
Vanilla Ice Cream            Negative           Negative
Vanilla Ice Cream            Positive           Positive
Milk                         Positive           Positive
Milk                         Negative           Negative
Raw Chicken(Deli)            Positive           Positive
Ground Turkey (cooked)       Negative           Negative
Ground Turkey (cooked)       Positive           Positive
Chilli Meat (Ground Beef)    Positive           Positive
Ground Beef Raw              Positive           Positive
Ground Beef (cooked)         Negative           Negative
Ground Beef(cooked)          Positive           Positive
Grated Cheese                Positive           Positive
Raw Turkey(ground)           Positive           Positive
--------------------------------------------------------------

References

Bagwell, CB, Munson, ME, Christensen, RL, Lovett, EJ (1994) A new homogeneous assay system for specific nucleic acid sequences: poly-dA and poly-A detection. Nucleic Acids Res. 22, 2424-2425.

Birch, DE, Kolmodin, L, Wong, J, Zangenberg, GA, Zoccoli, MA, McKinney, N, Young, KKY, Laird, WJ (1996) Simplified hot start PCR. Nature 381, 445-446.

Erlich, HA, Gelfand, D, Sninsky, JJ (1991) Recent advances in the polymerase chain reaction. Science 252, 1643-1651.

F rster, Vth (1948) Zwischenmolekulare energiewanderung und fluorezenz. Ann. Phys. (Liepzig) 2, 55-75.

Gelfand, DH, Holland, PM, Saiki, RK, Watson, RM (1993) U.S. Patent 5,210,015.

Higuchi, R, Dollinger, G, Walsh, PS, Griffith, R (1992) Simultaneous amplification and detection of specific DNA sequences. Bio/Technology 10, 413-417.

Higuchi, R, Fockler, C, Dollinger, G, Watson, R (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Bio/Technology 11, 1026-1030.

Holland, PM, Abramson, RD, Watson, R, Gelfand, DH (1991) Detection of specific polymerase chain reaction products by utilising the 5' to 3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. 88,7276-7280.

Lakowicz, JR (1983) Energy transfer. In Principles of fluorescent spectroscopy, Plenum Press, New York, NY, pp 303-339.

Lee, LG, Connell, CR, Bloch W (1993) Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res. 21, 3761-3766.

Livak, KJ, Flood, SAJ, Marmaro, J, Giusti,W, Deetz, K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridisation. PCR Meth Appl. 4, 357-362.

Livak, KJ, Marmaro, J, Todd, JA (1995) Towards fully automated genome-wide polymorphism screening. Nature Genetics. 9, 341-342.

Livak, KJ (1996) Quantitation of DNA/RNA using real-time PCR detection. P-E Applied Biosystems technology review, reference number 777902-001.

Lyamichev, V, Brow, MAD, Dahlberg, JE (1993) Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260, 778-783.

Stryer, L, Haugland, RP (1967) Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. 58,719-726.

Stryer, L (1978) Fluorescence energy transfer as a spectroscopic ruler. Ann. Rev. Biochem. 47,819-846.

Tyagi, S, Kramer, FR (1996) Molecular beacons: probes that fluoresce upon hybridisation. Nature Biotechnol. 14, 303-308.

Copyright 1996 Australian Biotechnology Association Ltd.


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