Abstract
The conference Applications of Nucleic Acids Technologies in Molecular Diagnostics (part of TIDES) consisted of four sessions: Regulatory Pathways and Quality Strategies, Manufacturing and Business Considerations for Successful Launch in the Clinical Market, Analytical Methods and Validation, and New Technologies. The conference brought together approximately 100 representatives from academia, clinical laboratories and industry and comprised 26 presentations. This article only focuses on new developments discussed in the session regarding New Technologies, with special emphasis on presentations highlighting real-time amplification and detection.
miRCURY LNA™ Universal RT miRNA PCR system
Thoraninn Blondal (Exiqon, Vedbaek, Denmark) reported on a new-generation high-throughput quantitative PCR platform optimized for development of miRNA-based molecular diagnostic assays on clinical formalin-fixed paraffin-embedded tissue, blood serum and plasma. miRNAs constitute a class of non-coding RNAs that play key roles in the regulation of gene expression. Acting at the post-transcriptional level, these molecules fine tune the expression of as much as 30% of all mammalian protein-encoding genes. Mature miRNAs are short, single-stranded RNA molecules approximately 22 nucleotides in length.
In addition to their important roles in healthy individuals, miRNAs have also been implicated in a number of diseases, including a broad range of cancers, heart disease and neurological diseases. Consequently, miRNAs are being intensely studied as candidates for diagnostic and prognostic biomarkers and predictors of drug response. The miRCURY Locked Nucleic Acid (LNA)™ Universal Reverse Transcription (RT) miRNA PCR system (Exiqon A/S, Vedbaek, Denmark) uses the well-known LNA technology enabling an improved mismatch discrimination. The system was successfully applied on RNA isolated from formalin-fixed paraffin-embedded tissue. A signature of four miRNAs able to separate normal colon from colon cancer was developed using this miRCURY LNA Universal RT miRNA PCR system Citation[1].
Universal transcription-mediated amplification
Norman Nelson (Gen-Probe, CA, USA) described the progress made on a multiplex format of the transcription-mediated amplification (TMA) technology. TMA is an RNA transcription amplification system using two enzymes to drive the reaction: RNA polymerase and reverse transcriptase. TMA is isothermal; the entire reaction is performed at the same temperature in a water bath or heat block. This is in contrast to other amplification reactions, such as PCR, which require a thermal cycler instrument to rapidly change the temperature to drive the reaction.
Transcription-mediated amplification can amplify either DNA or RNA and produces RNA amplicons, in contrast to most other nucleic acid amplification methods, which only produce DNA. TMA involves very rapid kinetics resulting in a billion-fold amplification within 15–30 min. The standard TMA cannot be used in a multiplex reaction owing to interference between the oligomers. The solution was the design of a reverse universal half TMA or a reverse universal full TMA. Both systems consist of a target capture oligomer, a blocker oligo and a directly hybridized complex of two oligomers containing the universal T7 and the universal non-T7 sequences. As a proof of principle, the system was used in a PCA3/PSA/internal control triplex assay for identification of prostate cancer. A good correlation was found between this triplex assay and the current CE-marked PROGENSA® PCA3 assay (Gen-Probe Inc., CA, USA).
Cold-PCR
Mike Makrigiorgus (Dana-Farber Cancer Institute, Harvard Medical School, MA USA) presented work on a new technology called co-amplification at lower denaturation temperature-PCR (Cold-PCR; Transgenomic Inc., NE, USA) Citation[2]. Cold-PCR is a modified PCR protocol that enriches variant alleles from a mixture of wild-type and mutation-containing DNA. Standard PCR will amplify both the major (wild-type) and minor (mutant) DNA with the same efficiency, occluding the ability to easily detect the presence of low-level mutations. The principle of Cold-PCR is as follows: a single nucleotide mismatch anywhere along a double-stranded DNA sequence generates a small but predictable change to the melting temperature (Tm) of DNA for that sequence. For each DNA sequence, there is a critical denaturation temperature (Tc) that is lower than the Tm. DNA amplicons differing by a single nucleotide have been found to result reproducibly in different amplification efficiencies when PCR denaturation is set to the Tc. This new observation can be exploited during PCR amplification for the selective enrichment of minority alleles differing by one or more nucleotides at any position of a given sequence. Cold-PCR run on a quantitative PCR machine has numerous applications in cancer diagnostics and personalized medicine. Additional areas that require detection of low-level mutations include infectious diseases, for the early detection of resistant strains emerging in a population of drug-responsive strains, and prenatal diagnosis, for detection of small amounts of fetal circulating DNA in the maternal blood.
Single-tube multiplex amplification in real time: SmartFinder technology
Guus Simons (PathoFinder, Maastricht, The Netherlands) described a new technology enabling the analysis of 19 different pathogens simultaneously on a real-time PCR machine. The SmartFinder technology is derived from the MultiFinder technology (PathoFinder) Citation[3], which uses two probes. One probe consists of a universal forward primer binding site and a target-specific sequence at the 3´ end, whereas the other probe consists of a target-specific sequence at the 5´ end, a stuffer sequence varying in size from 80 up to 400 nucleotides and a universal reverse primer binding site at the 3´ end. A MultiFinder assay involves ligation of the two probes using the oligonucleotide ligation assay, amplification with a universal set of PCR primers and, subsequently, the identification of the ligated probes, which is performed by size fractionation using a capillary electrophoresis system. Fluorescent-labeled hybridization probes are included in the SmartFinder assay (PathoFinder BV, Maastricht, The Netherlands) as well and detect the different MultiFinder probes using a real-time PCR machine. The combination of different fluorescent labels and specific Tms (from 50°C up to 80°C with 5°C intervals) enables the development of highly complex real-time PCR assays. Up to now, multiplex real-time PCR assays have been able to detect up to five targets in one reaction. The SmartFinder technology enables the simultaneous detection of 19 different causative agents (15 viruses among the recent swine flu, and four bacterial targets) of a respiratory tract infection. The technology is as sensitive as monoplex real-time PCR showing great potential in the fast and easy multiparameter screening of clinical samples for infectious agents.
Zip nucleic acids
Nathalie Lenne (Polyplus, Illkirch, France) reported about Zip nucleic acids (ZNAs) – high-affinity primers and probes as promising tools for molecular diagnostics Citation[4]. ZNAs are oligonucleotides conjugated with cationic spermine units that increase affinity for their target by decreasing electrostatic repulsion between negatively charged anionic single-strand nucleic acids to improve hybridization, thus enhancing and accelerating target recognition. The possibility of modulating the global charge of the ZNA oligonucleotide–oligocation conjugates by the number of cationic spermine moieties attached to the nucleic acid oligomer is key to easily predicting the Tm of ZNA–DNA/ZNA–RNA hybrids. The Tm increases linearly with the length of the oligocation. ZNAs were shown to enable specific and sensitive reactions when used as primers for PCR and RT. Moreover, ZNA probes provide broad flexibility in assay design and represent an effective alternative to minor groove binder- and LNA-containing oligonucleotides. In addition, efficient detection takes place without probe degradation and, hence, ZNAs can be considered as emergent and promising molecules for real-time PCR applications.
Hot Diamond Taq® polymerase
Marie-Claire Beckers (Eurogentec, Liege, Belgium) described a new Taq polymerase, called Hot Diamond Taq® (Eurogentec), for diagnostic PCR applications. The enzyme exhibits unique hot-start characteristics and represents a completely new ‘hot-start concept’. Hot-start characteristics are not accomplished through chemical modification nor a blocking antibody, but a proprietary agent prevents non-specific polymerization, thereby preventing primer-dimer formation and increasing the PCR yield of specific products. Hot Diamond Taq shows no amplification at room temperature and gives a very high yield of specific product. The enzyme needs very short activation time (100% are activated during the first PCR cycle) but is compatible with all existing protocols. This enzyme shows very good fidelity and catalyzes 5´→3´ synthesis of DNA with no detectable 3´→5´ proofreading exonuclease activity. Since it amplifies DNA templates at very low concentrations, this enzyme might be a good alternative for very sensitive real-time PCR applications.
Conclusion
The New Technology session of the Applications of Nucleic Acids Technologies in Molecular Diagnostics meeting demonstrated very interesting developments in real-time PCR. Real-time PCR is currently still the gold standard in molecular diagnostics of infectious diseases. It is expected that these new technologies, new primer and enzyme formulations will change the landscape towards multiplex analysis in a real-time format with the same sensitivity and specificity as, but being more cost effective than, monoplex assays for various applications in clinical diagnostic laboratories.
Financial & competing interests disclosure
The author is an employee of PathoFinder BV (discussed in the SmartFinder technology session) and was a presenter at this conference. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
References
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- Milbury C, Li J, Makrigiorgos G. PCR-based methods for the enrichment of minority alleles and mutations. Clin. Chem.55(4), 632–640 (2009).
- Reijans M, Dingemans G, Klaassen C et al. RespiFinder: a new multiparameter test to differentially identify fifteen respiratory viruses. J. Clin. Microbiol.46(4), 1232–1240 (2008).
- Moreau V, Voirin E, Paris C et al. Zip nucleic acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription. Nucleic Acids Res.37(19), e130 (2009).