Description
The development of the polymerase chain reaction, or PCR, by Saiki et al. (1) was a milestone in biotechnology and heralded the beginning of the modern era of molecu-lar diagnostics. Although PCR is the most widely used nu-cleic acid amplification strategy, other strategies have been developed, and several have clinical utility. These strate-gies are based on either signal or target amplification. Ex-amples of each category will be discussed in the sections that follow. These techniques have sensitivity unparalleled in laboratory medicine, have created new opportunities for the clinical laboratory to impact patient care, and have be-come the new “gold standards” for laboratory diagnosis of many infectious diseases.
SIGNAL AMPLIFICATION TECHNIQUES
In signal amplification methods, the concentration of the probe or target does not increase. The increased analytical sensitivity comes from increasing the concentration of la-beled molecules attached to the target nucleic acid. Multi-ple enzymes, multiple probes, multiple layers of probes, and reduction of background noise have all been used to en-hance target detection (2). Target amplification systems generally have greater analytical sensitivity than signal am-plification methods, but technological developments, par-ticularly in branched DNA (bDNA) assays, lowered the limits of detection to levels that rivaled those of some ear-lier target amplification assays (3).
Signal amplification assays have several advantages over target amplification assays. In signal amplification systems, the number of target molecules is not altered, and as a re-sult, the signal is directly proportional to the amount of the target sequence present in the clinical specimen. This reduces concerns about false-positive results due to cross-contamination and simplifies the development of quan-titative assays. Since signal amplification systems are not dependent on enzymatic processes to amplify the target sequence, they are not affected by the presence of enzyme inhibitors in clinical specimens. Consequently, less cumber-some nucleic acid extraction methods may be used. Typi-cally, signal amplification systems use either larger probes or more probes than target amplification systems and, conse-quently, are less susceptible to errors resulting from target se-quence heterogeneity. Finally, RNA levels can be measured directly without the synthesis of a cDNA intermediate.
bDNA
The bDNA signal amplification system is a solid-phase, sandwich hybridization assay incorporating multiple sets of synthetic oligonucleotide probes (4). The key to this tech-nology is the amplifier molecule, a bDNA molecule with 15 identical branches, each of which can bind to three la-beled probes.
The bDNA signal amplification system is illustrated in Fig. 1. Multiple target-specific probes are used to capture the target nucleic acid onto the surface of a microtiter well. A second set of target-specific probes also binds to the tar-get and to preamplifier molecules, which in turn bind to up to eight bDNA amplifiers. Three alkaline phosphatase-labeled probes hybridize to each branch of the amplifier. Detection of bound labeled probes is achieved by incubat-ing the complex with dioxetane, an enzyme-triggerable sub-strate, and measuring the light emission in a luminometer. The resulting signal is directly proportional to the quantity of the target in the sample. The quantity of the target in the sample is determined from an external standard curve.
Nonspecific hybridization of any of the amplification probes and nontarget nucleic acids leads to amplification of the background signal. To reduce potential hybridiza-tion to nontarget nucleic acids, isocytidine (isoC) and iso-guanosine (isoG) were incorporated into the preamplifier, and labeled probes were used in the third-generation bDNA assays (5). IsoC and isoG form base pairs with each other but not with any of the four naturally occurring bases (6).
The use of isoC- and isoG-containing probes in bDNA assays increases target-specific signal amplification without a concomitant increase in the background signal, thereby greatly enhancing the detection limits without loss of spec-ificity. The detection limit of the third-generation bDNA assay for human immunodeficiency virus type 1 (HIV-1) RNA is 75 copies/ml. bDNA assays for the quantification of hepatitis B virus DNA, hepatitis C virus (HCV) RNA, and HIV-1 RNA are commercially available (Siemens Healthcare Diagnostics, Deerfield, IL). The SiemensVersant 440 analyzer for bDNA assays automates the incubation, washing, reading, and data-processing steps.
Hybrid Capture
The hybrid capture system is a solution hybridization-antibody capture method that uses chemiluminescence detection of hybrid DNA-RNA duplexes (Fig. 2). The tar-get DNA in the specimen is denatured and then hybrid-ized with a specific RNA probe. The DNA-RNA hybrids are captured by antihybrid antibodies that are used to coat the surface of a tube. Alkaline phosphatase-conjugated anti-hybrid antibodies bind to the immobilized hybrids. The bound antibody conjugate is detected with a chemilumines-cent substrate, and the light emitted is measured in a lu-minometer. Multiple alkaline phosphatase conjugates bind to each hybrid molecule, amplifying the signal. The inten-sity of the emitted light is proportional to the amount of target DNA in the specimen. Hybrid capture assays for de-tection of Neisseria gonorrhoeae, Chlamydia trachomatis, and human papillomavirus in clinical specimens are available from Qiagen, Germantown, MD (7). There are manual and automated (rapid capture system) versions of these assays.
Cleavase-Invader Technology
Invader assays (Hologic/Gen-Probe, San Diego, CA) are based on a signal amplification method that relies upon the specific recognition and cleavage of particular DNA structures by cleavase, a member of the FEN-1 family of DNA polymerases. These polymerases will cleave the 5¢single-stranded flap of a branched base-paired duplex. This enzymatic activity likely plays an essential role in the elim-ination of the complex nucleic acid structures that arise during DNA replication and repair. Since these structures may occur anywhere in a replicating genome, the enzyme recognizes the molecular structure of the substrate without regard to the sequence of the nucleic acids making up the DNA complex (8, 9).
In the invader assays, two probes are designed which hybridize to the target sequence in an overlapping fashion (Fig. 3). Under the proper annealing conditions, the probe oligonucleotide binds to the target sequence. The invader oligonucleotide probe is designed such that it hybridizes upstream of the probe with a region of overlap between the 3¢ end of the invader and the 5¢ end of the probe. Cleavase cleaves the 5¢ end of the probe and releases it. It is in this way that the target sequence acts as a scaffold upon which the proper DNA structure can form. Since the DNA structure necessary to serve as a cleavase substrate will occur only in the presence of the target sequence, the generation of cleavage products indicates the presence of the target. Use of a thermostable cleavase enzyme allows reactions to be run at temperatures high enough for a pri-mer exchange equilibrium to exist. This allows multiple cleavase products to form off of a single target molecule. FRET probes and a second invasive cleavage reaction are used to detect the target-specific products. FDA-cleared as-says for detection of pools of high-risk genotypes and types 16 and 18 of human papillomavirus in cervical samples are available from Hologic/Gen-Probe (10, 11).