Medical Device Link.

Originally Published IVD Technology November/December 2005

MOLECULAR DIAGNOSTICS

Developments in PCR detection methods

Homogeneous detection technologies improve performance in PCR-based diagnostics.

Stephen Little

A 96-head pipetting arm from the main automated workstation by DxS Ltd.

Molecular diagnostics is the fastest-growing segment of the IVD market. In 2002, 30 million to 40 million molecular tests were performed in the United States alone. Even though the molecular diagnostics segment currently accounts for only 5% of the IVD market, it is expected to grow rapidly as a result of the high degree of innovation in this area by the IVD industry. By 2010, revenues in the molecular diagnostics market are expected to reach $3.7 billion, representing an annual growth rate in excess of 20%.

The short-term factors driving this expected 20%-plus growth include blood donor screening, human papilloma virus (HPV) testing, chlamydia and gonorrhea testing, and viral load testing. Long term growth drivers are expected to be the identification of new genes associated with specific diseases, the identification of single nucleotide polymorphisms (SNPs) associated with responses to certain drugs, and the development of more-automated nucleic acid testing platforms.

Since its invention in 1986, the polymerase chain reaction (PCR) has been a cornerstone of molecular biology, underpinning many of the field’s most significant discoveries.¹ Such breakthroughs include the identification of genes responsible for many genetic diseases, the successful completion of the Human Genome Project, and the elucidation of the nucleotide sequence of numerous important organisms.¹

IVD manufacturers have been using PCR widely for developing molecular diagnostic technologies. Many of the PCR-based products are designed to detect infectious diseases, while other products detect and enumerate bacteria and other microorganisms. Recently, test kits have become available for detecting inherited and acquired human genetic variations.

PCR Domination

To some extent, the development of the PCR-based diagnostics market has been hindered by Roche Diagnostics’ (Basel, Switzerland) possession of PCR patents. By owning such patents, Roche has maintained a significant level of control over the development of the PCR market. Given this situation, which has existed since the late 1980s, it is hardly surprising that many IVD companies have attempted to invent and develop alternatives to PCR.

Figure 1. The Scorpions reaction. Scorpions primers come in two formats: closed (shown above) in which the fluorophore and quencher are held together by an internal stem loop, or open, in which the quencher is supplied on a separate oligonucleotide. In steps 1 and 2, the Scorpions primer is extended on the target DNA. In step 3, the extended primer is heat denatured, and the quencher disassociates. In step 4, the extended Scorpions primer is rearranged as it cools and begins to fluoresce in a target-specific manner, while the unextended primer is quenched.

Such novel alternative PCR methods include nucleic acid sequence–based amplification (NASBA), branched DNA, strand displacement amplification, and the loop-mediated isothermal amplification (LAMP) method owned by Sysmex Corp. (Kobe, Japan).^2-5 For nearly 20 years, IVD manufacturers have made significant and sustained efforts to develop PCR alternatives. While many of the novel approaches are functional, none of them exhibit the specificity and simplicity of the original PCR invention. Consequently, PCR has dominated the molecular diagnostics market, and its demonstrated utility in diverse applications suggests that it will continue to enjoy its top position.

Earlier this year, a significant development took place which is likely to reinforce PCR’s dominance in the molecular diagnostics market. In March, the foundational PCR process patents expired in the United States; they will expire one year later in the rest of the world. Since PCR has become the leading molecular diagnostics technique despite Roche’s exclusive patent rights, lifting this intellectual property burden is likely to ensure PCR’s continued supremacy in the molecular diagnostics market.

However, not all or even most of the PCR patents have expired. Roche currently holds more than 300 PCR-related patents in the United States. Among those, only the following patents have expired: process for amplifying, detecting, and/or cloning nucleic acid sequences; process for amplifying nucleic acid sequences; process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme; kits for amplifying and detecting nucleic acid sequences including a probe; kits for amplifying and detecting nucleic acid sequences; detection of AIDS-associated virus by PCR; and detection of viruses by amplification and hybridization.

Although these patents are only a small fraction of the total, they are very significant because they cover the basic PCR amplification processes, and their expiration will open up the PCR-based diagnostics market. All molecular diagnostics are comprised of at least three sample-preparation steps, followed by amplification and detection. With basic PCR amplification being more readily available, IVD manufacturers have shifted their focus to PCR detection methods as the second key technological requirement for PCR-based diagnostics.

PCR Detection

As the molecular diagnostics market has matured, IVD manufacturers have gained a better understanding of which assay formats are appropriate for different applications. The assays that are on the market and those that are being developed can be classified into four groups, based on whether they are qualitative or quantitative, and whether they are low complexity (one or two analytes) or high complexity (several analytes).

Figure 2. Quantitative PCR using Scorpions primers to detect the ß-actin gene in genomic DNA. The first graph shows real-time PCR traces indicating the amount of input genomes (a). The second graph shows the relationship between input DNA and threshold cycle (b).

For the qualitative assays, the range of detection methods includes colorimetric and size-based techniques. Quantitative assays rely on homogeneous fluorescent detection methods to allow quantitation by real-time PCR. Homogeneous fluorescent methods can also be applied to qualitative assays.

Two types of detection methods for PCR products are available. Heterogeneous detection methods require a PCR vessel to be opened after a reaction is completed to allow manipulation of a PCR product. Examples of heterogeneous methods include gel electrophoresis or DNA microarray hybridization. In homogeneous or closed-tube detection methods, all of the reactants are placed in a PCR vessel prior to initiating a reaction, and the presence of a PCR product is detected without opening a tube. This is normally done by monitoring an increase in fluorescence of a PCR reaction.

Newly emerging nucleic acid–based diagnostics are increasingly moving toward more-complex assays requiring simultaneous detection of many different genetic markers. For qualitative assays (e.g., multiplex analysis of many SNPs), array-based methods using DNA chips, beads, or fiber-optic cables are emerging as the de facto standard for accurate quantitation; however, real-time PCR is needed, and homogeneous fluorescent detection methods dominate. For quantitative analysis of several analytes, it is often useful to multiplex assays together, which requires a range of nonoverlapping fluorescent dyes and compatible detection instruments.

Based on this, IVD manufacturers will increasingly require access to homogeneous fluorescent detection methods that allow assay quantitation by real-time monitoring of PCR and simplify signal detection of qualitative assays.

Even though Roche owns one of the most important homogeneous detection methods, the 5' nuclease assay or Taqman, its patents do not claim the concept of homogeneous nucleic acid detection. Several detection alternatives are on the market, some of which offer performance benefits over the Taqman technology while maintaining compatibility with real-time PCR instrumentation.¹⁰

Homogeneous PCR Detection Methods

The various homogeneous detection technologies can be classified into two basic types: nonspecific and specific. Nonspecific methods signal that PCR has taken place within a reaction vessel, while specific methods signal that PCR has taken place and a particular product has been produced (see Table I).

In general, nonspecific homogeneous detection methods are not suitable for diagnostic applications because of the potential for false positives caused by primer dimers and other nonspecific amplicons. In contrast, with the exception of HyBeacons, all of the specific detection methods are suitable for real-time PCR and potentially available to IVD manufacturers. The Scorpions technology from DxS Ltd. (Manchester, UK), for example, is a specific detection method in which signal generation is through a unimolecular rearrangement rather than a bi-, or tri-, molecular collision.

The Scorpions Technology

Figure 3. Direct comparison of three real-time PCR detection systems.

The Scorpions technology features bifunctional molecules containing a PCR primer covalently linked to a probe. The fluorophore in the probe interacts with a quencher that reduces fluorescence. During a PCR reaction, the fluorophore and quencher are separated, which leads to an increase in light output from the reaction tube (see Figure 1).

An example of performance data shows detection of the ß-actin gene in dilutions of genomic DNA (see Figure 2). This assay detects as few as two copies of input DNA and demonstrates good correlation between input material and threshold cycle. As with all fluorescent detection systems, the ability to multiplex Scorpions reactions is limited by the number of available fluorescent dyes and instrumentation. At present, four separate Scorpions reactions can be performed in a single assay. Assay design software is also available for Scorpions primers, and using this tool ensures that assay design success rates are high. To date, all targets tested have resulted in successful designs, which include real-time quantitation assays, endpoint genotyping assays, and quantitative mutation assays for oncology and infectious-disease testing.

The difference between the Scorpions technology and other homogeneous detection systems is that in Scorpions reactions, the probe and the target are in the same molecule so that signal generation is through a unimolecular rearrangement. In contrast, all other detection systems require a bimolecular collision. This difference means that the kinetics of signal generation in Scorpions reactions are rapid.

Technical Performance

Signal-to-Noise Ratio. The Scorpions technology’s unimolecular rearrangement is a fast and efficient reaction. A typical real-time experiment compares the performance of three detection systems: Scorpions, Taqman, and Molecular Beacons (see Figure 3). While the probe sequence is identical in all three systems, it is covalently linked to the primer molecule in the Scorpions reaction. The data show that while the PCR efficiency is similar in all three systems (the threshold cycle is the same in all three reactions), the Scorpions technology generates about twice the increase in fluorescence of Taqman because of a more efficient probing reaction.

Figure 4. Real-time PCR of dilutions of Bacillus spp DNA detected using a Scorpions primer (a). Real-time PCR data showing the relationship between threshold cycle and input DNA, and the absolute time to detection (b).

Fast PCR. For some applications (e.g., detection of biothreat agents, intra-operative diagnostic testing), rapid PCR is a benefit. The two requirements for rapid real-time PCR are a thermal cycler that allows rapid heating, cooling, and thermal transfer, and a signal generation system that is compatible with the short cycle times associated with fast PCR. Many of the specific real-time detection systems take several seconds to generate a signal (see Table I). While this is not a problem with normal PCR, it can limit the maximum speed of fast PCR if the cycle times need to be extended to allow a probe to generate fluorescence.

Figure 5. Single-tube end point genotype assay for UGT1A1 alleles *1 (6AT) and *28 (7AT) for 96 unrelated Caucasian individuals. The two Scorpions are labelled with different dyes, and the ratio of the two dyes indicates the genotype.

Because the method of signal generation in a Scorpions reaction is so fast, it is possible to perform rapid PCR reactions without losing efficient signal generation. The rate of signal accumulation in real-time Scorpions and Taqman reactions can be compared. The reactions were performed in a LightCycler instrument, which allows rapid cycling. The PCR parameters were denaturation 94°C for 1 second, extension 60°C for 120 seconds. In the Taqman reaction, the accumulation of fluorescence as the probe is cleaved during each PCR cycle takes 20–40 seconds. In contrast, with the Scorpions reaction, signal generation is instantaneous. The Scorpions reaction that is extended in the previous cycle undergoes an internal rearrangement, and as soon as the extension temperature is reached, the signal is generated.

In a standard PCR reaction with slow cycling conditions, this signal generation difference is not significant. However, when the extension times are reduced in rapid PCR, this feature becomes an advantage and translates into fast PCR. With the appropriate instrumentation, single-copy bacterial sequences can be detected in less than 15 minutes. One example is the rapid detection of low levels of Bacillus spp using Scorpions primers and a fast PCR machine (see Figure 4). In addition to the real-time traces, the threshold cycle values have been taken and replotted to show time to detection. The higher levels of input DNA were detected in less than 10 minutes, and even low levels could be detected in less than 14 minutes.

Stephen Little, PhD, is chief executive officer at DxS Ltd. (Manchester, UK). He can be reached at stephen.little@ dxsgenotyping.com.

Melting Temperature Bonus and Short Probes. Another feature of the Scorpions technology’s unimolecular mode of action is the melting temperature (Tm) bonus. This feature refers to the fact that the Tm for a Scorpions probe, which is in the same molecule as its target, can be up to 300°C higher than the same probe and target sequences in different molecules. The benefit of the Tm bonus for a molecular diagnostic product is that a shorter probe can be used to detect a target compared with a bimolecular system. This is important for SNP detection because the effect on hybridization with a mismatched SNP is more destabilizing with a short probe, and hence there is better discrimination between the different alleles. For example, a Scorpions assay for the UGT1A1*28 allele contains a run of 7AT repeats and differs from the 6AT*1 wild type sequence (see Figure 5). The discrimination due to the Tm bonus allows a reliable test for this difficult genetic polymorphism.

Conclusion

The Scorpions technology has an intellectual property position that is independent from Taqman and all other homogeneous detection systems. For IVD manufacturers, the use of Scorpions or one of the alternative detection systems does not remove the need to obtain a license from the patent holder. However, the existence of a number of different detection methods means that there will be a healthy level of competition in the IVD marketplace, leading to easier access to and a greater range of molecular diagnostic products in the future.

References

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