ReviewCombining nucleic acid amplification and detection
Introduction
Molecular amplification methods are fundamental to laboratory research, pharmacogenomics and molecular diagnostics. They fall into two classes, enabling either target or signal amplification (Table 1). Target amplification methods include polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), and nucleic acid sequence-based amplification (NASBA). All are very sensitive and compatible with many detection techniques, such as fluorescence, chemiluminescence, or gel electrophoresis. Signal amplification technologies include branched DNA (bDNA), hybrid capture, and cleavase, and measure nucleic acid targets by amplification of a surrogate marker. Rolling circle amplification (RCA) is a newer method that performs either target or signal amplification.
The aim of this review is to highlight advances in the use of these technologies during the past year. In addition, novel procedures for detecting the products of amplification reactions are discussed.
Section snippets
Polymerase chain reaction
PCR remains the most widely used DNA amplification method. Food and Drug Administration (FDA)-approved diagnostic kits (Roche) that use PCR for the detection and quantitation of HIV, Mycobacterium tuberculosis, and Chlamydia trachomatis were the subject of numerous studies during the past year that demonstrated clinical accuracies equivalent or superior to other nucleic acid detection techniques, such as bDNA and NASAB [1], [2], [3], [4].
A significant recent advance in PCR was the development
Ligase chain reaction
In LCR, two contiguous oligonucleotides are joined by DNA ligase upon perfect hybridization to a DNA target. Ligated probes are amplified by thermal cycling with complementary oligonucleotides [16]. During the past year, several studies of an FDA-approved LCR diagnostic test for C. trachomatis infection showed comparable clinical sensitivity to PCR [17].
LCR has better allele specificity than PCR for genotyping point mutations and SNPs because of the greater discriminatory power of ligation over
Strand displacement amplification
The mechanism of amplification by SDA is shown in Fig. 2. Amplified targets are detected by inclusion of a single-stranded probe containing two different fluors. Upon SDA, the probe is incorporated into a double-stranded product that is cleaved by a restriction endonuclease, eliminating fluorescence quenching. A semi-automated system (ProbeTecET, Becton-Dickinson) for carrying out SDA with real-time fluorescence detection was recently introduced, which may reinvigorate interest in this method
Nucleic acid sequence-based amplification
NASBA is commonly used for measuring HIV viral load (Organon Teknika). NASBA amplifies RNA and DNA targets as antisense, single-stranded RNA by the concurrent activity of reverse transcriptase, RNase H, and T7 RNA polymerase and two primers [24].
Recent comparisons of HIV-1 assays by NASBA and other methods differed in their conclusions [1], [2], [25], [26], indicating a need for further evaluation of NASBA. This past year also saw the launch of a NASBA-based general RNA assay (Qiagen).
Transcription-mediated amplification
Transcription-mediated amplification (TMA) is an RNA transcription amplification method that uses RNA polymerase and reverse transcriptase in an isothermal reaction to amplify either DNA or RNA targets. In the TMA assay (Gen-Probe Inc., San Diego, CA, USA), amplicons are detected homogenously by chemiluminescence.
TMA is FDA-approved for detection of C. trachomatis and M. tuberculosis. A TMA assay for quantification of HIV-1 RNA was recently shown to be more sensitive than reverseRNA was
Branched DNA
bDNA achieves signal amplification by attaching many alkaline phosphatase molecules to a DNA dendrimer. Several tree-like structures are built in each molecular recognition event. The Quantiplex bDNA assay (Chiron) uses a dioxetane substrate for alkaline phosphatase to produce chemiluminescence. Although RT-PCR is the only FDA-approved method for quantitation of HIV-1 RNA in plasma, bDNA is frequently used in clinical laboratories. An improved bDNA test was recently shown to be linear from
Hybrid capture
In Hybrid Capture (Digene), target DNA molecules hybridize to specific RNA probes. RNA–DNA hybrids are captured on a solid phase coated with specific antibodies and detected with a chemiluminescent substrate using antibodies conjugated with alkaline phosphatase.
The system has been approved by the FDA for the detection of N. gonorrhoeae, C. trachomatis, and cytomegalovirus. Hybrid Capture is the only FDA-approved DNA-based assay for the detection of human papillomavirus (HPV) [33]; this year,
DNA cleavage-based signal amplification
Two signal amplification methods based on DNA cleavage have been described. In cycling probe technology (CPT; ID Biomedical), a chimeric DNA–RNA probe is cleaved by RNAse H upon target binding. Probe cleavage is detected by either immunoassay on a lateral flow device or colorimetry. Two recent studies reported the use of CPT for diagnosis of methicillin-resistant Staphylococcus aureus [34] and vancomycin-resistant enterococci [35].
Invader (Third Wave) is the second cleavage-based assay. Invader
Rolling circle amplification
RCA is a new amplification method that is useful both for signal and target amplification [39]. Linear and exponential forms of RCA have been described. In linear RCA, a DNA circle is amplified by polymerase extension of a complementary primer. Up to 105 tandemly repeated, concatemerized copies of the DNA circle are generated by each primer (Fig. 3). Target amplification by linear RCA is limited to circular nucleic acids, such as circular viruses, plasmids, and circular chromosomes. Using
Nanoparticles
Many DNA sequence detection applications utilize hybridization of labeled oligonucleotides. A novel detection scheme based on nanoparticles was recently described that uses oligonucleotides covalently bound to gold particles [43].
One benefit of using nanoparticle labels was alteration of the melting profile of the labeled probe to improve discrimination of single base mismatches by hybridization. In addition, sensitivity was increased 100-fold compared with fluorescence by signal amplification
Conclusions
The past year has seen substantial development in new technologies for DNA amplification and detection as well as increased diversification in the ways various amplification methods are applied. PCR remains the most widely used method for DNA amplification for both research as well as diagnostic applications. With regard to the latter, PCR still faces competition from other technologies such as LCR, SDA, NASBA, TMA, bDNA, and hybrid capture, which, for the most part, show comparable clinical
References and recommended reading
Papers of particular interest, published within the annual period of review,have been highlighted as:
of special interest
of outstanding interest
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