advances in nucleic acid-based detection methodst · characteristics ofprobe andtarget nucleic...

CLINICAL MICROBIOLOGY REVIEWS, OCt. 1992, p. 370-386 Vol. 5, No. 40893-8512/92/040370-17$02.00/0Copyright X 1992, American Society for Microbiology

Advances in Nucleic Acid-Based Detection MethodstMARK J. WOLCOT[

Department ofMicrobiology, Immunology and Preventive Medicine, Iowa State University,Ames, Iowa 50011-3211

INTRODUCTION ................................................... 370THEORY OF NUCLEIC ACID-BASED DETECTION METHODS ........................................... .......370

Principles of Nucleic Acid Hybridization ................................................... 370Characteristics of Probe and Target Nucleic Acids .................................................... 371Probe form ................................................... 371Probe size................................................... 371Specificity of the probe ................................................... 371Labeling probes .................................................... 371Choice of target nucleic acids................................................... 371

HYBRIDIZATION STRATEGIES ................................................... 372Filter Hybridizations ................................................... 372Solution or Liquid Hybridizations ................................................... 372

AMPLIFICATION METHODS.................................................... 373Polymerase Chain Reaction ................................................... 374Ligase Amplification Reaction ................................................... 376Transcription-Based Amplification Systems................................................... 377Cycling Probe Reaction .................................................... 379Qf8Replicase Amplification .................................................... 379Strand Displacement Amplification ................................................... 380

SIGNAL AMPLIFICATION AND DETECTION METHODS................................................... 381AUTOMATION................................................... 383SUMMARY AND CONCLUSIONS................................................... 383ACKNOWLEDGMENTS .................................................... 384REFERENCES ................................................... 384

INTRODUCTION

Clinical microbiologists have traditionally been concernedwith the isolation and identification of pathogenic organismsfrom humans. Conventional methods involve isolating theorganism of interest in pure culture and performing prede-termined biochemical or immunologic tests to identify it.In many respects, cultures and the adjunct methods usedfor identification are limited in sensitivity, specificity, or

both. To improve test sensitivity, shorten detection times,and identify hard-to-culture microorganisms, immunoassayswere developed. These immunoassays allowed both largeand small laboratories to expand services to meet diagnosticrequirements in a timely fashion. The outlook for even more

sensitive, more specific, and more rapid testing is currentlybeing founded in the recent advances in molecular biologymethods.

Histologists, cytologists, and cytogeneticists have beenconcerned with the preparation and staining of various tissuesections for microscopic examination. The traditional stainshave allowed differentiation of various cellular components.Over the years, special stains to improve the detection ofnumerous cellular components and infectious agents ofinterest have been developed. This area of laboratory prac-tice was one of the first areas to benefit from the advances inmolecular biology over the past decade.

Nucleic acid analysis has attracted much attention over

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the past few years and is now becoming available for clinicallaboratory testing on a routine basis (105). The advantages ofnucleic acid probes in identifying organisms without specialor tedious isolation, detecting nonviable organisms, anddetecting genetic mutations or alterations in other cellularmaterial represent the promise of nucleic acid-based tech-nology (105). This review focuses on some methods fordetecting nucleic acid molecules and presents some newerand more novel methods that are being developed forpossible introduction into the clinical laboratory.

THEORY OF NUCLEIC ACID-BASED DETECTIONMETHODS

Principles of Nucleic Acid HybridizationIn 1953, Watson and Crick described the structure ofDNA

and the role of the DNA molecule in holding the informationof cell reproduction and function (100). DNA is composed offour repeating nucleotides (sometimes called nucleotidebases or simply bases): adenine, guanine, cytosine, andthymine. DNA is coiled to form a double helix (double-stranded DNA [dsDNA]) composed of two strands heldtogether by hydrogen bonds that can be broken by heat or

high pH. The single strands of DNA (ssDNA) are relativelystable, but on removal of the heat source or pH extreme, theDNA molecule will re-form (reanneal) into the double-stranded configuration. When the ssDNAs are from differentsources, the reannealing process is called hybridization. Therealigning of the dsDNA is possible because nucleotidebases will re-form hydrogen bonds only with specific com-

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NUCLEIC ACID-BASED DETECTION METHODS 371

plementary bases; adenine pairs with thymine, and cytosinepairs with guanine. In RNA, the nucleotide base uracilreplaces thymine and pairs with adenine. The stability of thehybridization depends on the nucleotide sequences of bothstrands. A perfect match in the sequence of nucleotidesproduces a very stable dsDNA, whereas one or more basemismatches impart increasing instability that can lead toweak hybridization of strands. The same basic principlesapply to RNAs, the transcriptional products of cell growthand reproduction. RNA-RNA and RNA-DNA hybridiza-tions can occur.

Characteristics of Probe and Target Nucleic Acids

Probe form. A probe is normally a short sequence ofnucleotide bases that will bind to specific regions of a targetsequence of nucleotides. The degree of homology betweentarget and probe results in stable hybridization. In develop-ing a probe, a sequence of nucleotides must be identified,isolated, reproduced in sufficient quantity, and tagged with acompound (label) that can be detected (89). Most often, aDNA sequence is cloned (inserted) into a host organism thatwill reproduce the sequence that becomes the probe. An-other method of producing probe sequences is by manufac-turing a synthetic oligonucleotide, which usually consists ofa short piece of nucleic acid composed of 15 to 30 nucleo-tides. The ability to produce on demand a synthetic piece ofnucleic acid that will function as a probe has allowed theeconomical commercial development of many probe sys-tems. Although manufactured probes require a detailedknowledge of the target sequence to produce a complemen-tary DNA molecule that will hybridize specifically, theiradvantages (single strandedness, noncomplementarity) favortheir use over cloned probes. It is beyond the scope of thispaper to discuss genetics and DNA cloning or synthesis, andreviews by Haas (35), Hilborne and Grody (37), Mulcahy(69), and Tenover (88) are recommended.An ideal probe is single-stranded nucleic acid that can

hybridize to the target of concern. The probe is composed ofeither DNA or RNA, with DNA probes being more common(38, 99). In general, RNA and DNA probes behave in thesame manner; therefore, references to probes in the follow-ing discussion include both DNA and RNA probes. Thesuitability of a probe for microbial detection is based onwhether a single strain or a broad family of organisms is ofinterest and whether the probe will detect other organismsthat are present (28, 76). Berry and Peter (7) adroitlysummarized the basic principles of hybridization and theapplication of probes: "Hence, the whole nature of anorganism is ultimately related to the sequence of nucleotidebases in its DNA. Not only can an organism be identified byits DNA sequence, but even minor differences betweensimilar organisms are detectable if the areas of nucleotidesequence responsible for these differences can be observed.This is exactly what a hybridization probe does."Probe size. Probes can range in size from as short as 10

nucleotide bases (molecular weight of 3,300) to as long as10,000 bases or more (molecular weight of 3,300,000) (30, 37,81). The most common size range for most probes is between14 and 40 bases (89). For statistical uniqueness, a minimumof 20 nucleotide bases are usually needed for a probe (7). AnEscherichia coli DNA chromosome, in contrast, is com-posed of about 4.2 x 106 bp. Except for probes that haveextra bases for the label, the entire length of the probenormally constitutes the combining site and affects thespecificity and sensitivity of the test. Short probes tend to

hybridize nucleic acids at very high rates (in minutes),whereas longer probes may require reaction times of hoursto achieve a stable hybridization (13, 89). However, shortprobes do have some disadvantages. They are subject tomore nonspecific hybridizations, are limited in specificity,and are more difficult to label (see the section on labelingprobes, below). Long probes hybridize more stably thanshort probes at high temperatures and low salt concentra-tions (low stringency). The advantages of rapid hybridiza-tion, longer stability, ease of preparation, and the ability todetect minor changes in the target nucleic acid by the shorterprobes (13, 89, 99) favor their use in most commercialassays.

Specificity of the probe. The base sequence of the probeand the conditions under which the probe is used determineits specificity (49). It is not necessary to know the function ofthe target nucleic acid or even the identity of the target orprobe sequence before a probe can be used (49). The onlyrequirements are that the probe hybridize specifically to thetarget nucleic acids and that the target nucleic acids beunique. Of course, the probe must not hybridize to itself orto nontarget nucleic acids in either the indigenous microbialflora or the clinical specimen (49).

Labeling probes. To be useful, probes must be detectableafter hybridization with the target. Like current clinicalimmunoassays, most probes are labeled so that they can bedetected after they hybridize. Labels can be either attachedor incorporated into the probe molecule. Isotopes such as32p, 35S, and 125I are often incorporated into the structure ofthe molecule. Enzymes such as alkaline phosphatase orhorseradish peroxidase are often covalently linked to theprobe (28, 55). The attachment of an enzyme via a covalentcross-linkage intermediate (41) is a variation of directlylinking enzymes to the nucleic acid molecule. Covalentlinkage of the label includes an intermediate compound (orspacer) that extends the enzyme away from the probe. Thisallows hybridization reactions to proceed without sterichindrance and permits full functionality of the enzyme label.Another variation of labeling nucleic acids involves incorpo-rating biotin into the probe and subsequently detecting thebiotin with enzyme-labeled avidin molecules. Biotin can beincorporated into the nucleic acid through a biotinylatednucleotide analog (a molecule similar to one of the fournucleotides) or through a photoactivated compound calledphotobiotin (28, 58, 69, 74, 89).

Similar to early immunoassay technology, the first nucleicacid hybridization studies used radioactive material forlabeling nucleic acid probes. The short half-life of most ofthe radioactive materials (14 days for 32p; 60 days for "2I)and the special handling and disposal requirements makethem unsuitable for most clinical laboratories and commer-cial kit manufacturers. Zeph et al. (106) compared a radio-actively labeled probe with three nonradioactively labeledprobes; the radioactively labeled probe was more sensitivebut produced more false-positive reactions than the nonra-dioactively labeled probes. The authors concluded, how-ever, that on the basis of ease of use, expense, and otherfactors, the nonradioactively labeled probes "were generallysuperior to the radiolabeled probe" (106).

Choice of target nucleic acids. The choice of targets forprobes should be of interest to clinical laboratory personnelin evaluating probe kits for possible use in their laboratory.Probes can be designed to detect genera, species, or, some-times, strains of various organisms, depending on the ex-pected target of hybridization. Another type of probe detectsa conserved gene (one that is the same in several genera or

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species) or a portion of it. Conserved regions of genesusually include portions of rRNA that represent a broadgroup or family of organisms. Even if the probe is targeted toa portion of nucleotide sequence that is present in microor-ganisms other than the one of interest, the probe will still besufficiently specific to be useful if the cells not of interest areusually absent from the sample. The use of rRNA nucleotidesequences, such as one for members of the family Entero-bacteniaceae, would be very useful in screening clinicalspecimens before genus-specific probes are used to identifythe exact pathogen (11).

Targets in cellular DNA are the most specific because theycontain unique regions. However, targets in RNA are alsoused because there are many more copies of RNA than DNAper cell, which increases the sensitivity of detection. Al-though mRNA is a possible target source, it is rarely usedowing to the often rapid turnover of the molecule in the cell(101). However, this disadvantage of mRNA targets can bean advantage when it is important to distinguish betweendormant and actively growing cells. rRNA is the most usefulprobe target in a screening assay. The 16S and 23S ribosomalgenes are very useful for detecting taxonomic groups be-cause of the highly conserved nature of the genes (47, 48).rRNA is more highly conserved than mRNA, and manymore copies of rRNA than DNA exist in each cell. E. coli,for instance, contains about 10,000 ribosomes per cell com-pared with only one or at most four copies of genomic DNAduring active growth. Thus, the detection of rRNA ratherthan DNA increases the sensitivity 1,000-fold or more (50).Other targets must be used for viruses, because viruses lackrRNA. Plasmid DNA (DNA that exists as extrachromo-somal elements) is an effective target in some microorgan-isms. The limitation of plasmid DNA, however, is thatplasmids are transferred between some organisms and maybe present in nontarget strains or lost from the cell alto-gether. Be it RNA or DNA, the best target is a sequence ofnucleotides (e.g., virulence factors, toxin genes) not presentin nontarget cells (28).

HYBRIDIZATION STRATEGIES

Like immunoassays, probes can be used in several differ-ent formats. These formats are generally classified into thosethat employ a solid phase and those that use a liquid phasefor the hybridization reaction. An easy-to-use format is alarge factor in determining the commercial success of vari-ous manufacturers' probe systems. This section describesvarious formats that are available.

Filter Hybridizations

Solid-phase, or filter, hybridization is usually in the formof dot or colony blots (32). The basic procedure for filterhybridization is shown in Fig. 1. This format is the oldest andmost often used in the research laboratory, but it is subjectto more nonspecific background interference than some ofthe other hybridization formats.To decrease the nonspecific background interference,

methods to remove probe that is not specifically bound to thesolid phase are being developed. The sandwich hybridizationassay (80) employs a three-component system consistin of asolid-phase bound capture probe, the target probe, and aseparate signal-generating probe (Fig. 2). The target probeacts as the link to the solid-phase and signal-generatingprobes. If the target probe is not present, the signal-gener-ating probe does not bind to the solid phase, and on washing,

no signal is generated; background interference is mini-mized.One variation of the solid-phase format is the strand

displacement assay (25, 96, 99). In this format (Fig. 3), thecapture probe is attached to a solid phase and has a shortsignal-bearing nucleic acid strand weakly hybridized to it.On addition of the target sequence, the target, throughcompetition, displaces the single-bearing strand. In oneapplication (96), the signal-bearing strand, which contained apolyadenylated signal, was detected by conversion to ATP,which was then detectable in a bioluminescence assay byusing luciferase. Although designed as a way of increasingsensitivity by decreasing background noise, this format doeshave shortcomings. The signal-bearing probe is not alwayseasily displaced by the target DNA, and thus it can compet-itively block hybridization of the capture probe with thetarget. Further, the signal-bearing strands are continuouslyleached from the capture probe, and this can lead to eitherfalse-positive reactions or high background.

Reversible target capture is a solid-phase format thatoffers a distinct advantage over other formats in that it canbe used with crude preparations without the generation ofbackground noise that can lead to false-positive reactions(67, 68, 77). Magnetic microparticles are used to separate thereactants from the cell lysates. The magnetic particles con-tain an oligonucleotide composed of a single nucleotide suchas poly(dA). This nucleotide will bind strongly to its com-plementary nucleotide, poly(dT). If a probe has a poly(dT)tail, the tail will be captured by the poly(dA) on the magneticparticle. The magnetic particle is removed from solution andwashed free of contaminating materials. Chemical or thermalelution of the dA-dT pair and its recapture on fresh micro-particles a few times leads to an exceedingly clean solidphase and increased sensitivity (67, 68, 77).A variation on the reversible target capture format is the

dipstick format (46, 77) (Fig. 4). As in reversible targetcapture, the probe has a long stretch of a single nucleotideand the dipstick has been coated with the complementarynucleotide. Because a second probe that has been labeledwith a detectable ligand is used, this format becomes veryversatile both in cleaning up the reactants and in allowingdetection. In one commercial application, fluorescein is thelabel on the probe. It is detected colorimetrically with ahorseradish peroxidase-labeled antifluorescein antibody andhydrogen peroxide-tetramethyl benzidine substrate-chro-mogen (46).

Solution or Liquid Hybridizations

Solution hybridization is more rapid (5 to 10 times faster)than hybridization on solid supports, but unless the assay isa homogeneous assay, a separation step is required beforefinal detection (89, 99). In addition, accelerators can beadded to increase the rate of hybridization even further;these are removed later to prevent background interference.Several separation steps, including the dipstick assay men-tioned above, can be used.

Hydroxyapatite binds only dsDNA. This means that ssDNAmust be hybridized either with a probe or with a complemen-tary strand before it can bind to hydroxyapatite. When asolution of DNA is passed through a hydroxyapatite column,only the dsDNA is retained. The hybridization mixture can bewashed to remove nonhybridized components (backgroundnoise) before being eluted from the column and before the labelis activated.Methidium, a nucleic acid intercalator dye that is coupled

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ISOLATE CELLS ON A SOLIDSUPPORT

CONVERT dsDNA TO ssDNA ANDBIND TO SOLID SUPPORT

HYBRIDIZE PROBE TO TARGET

DISRUPT CELLS TO OBTAIN dsDNA

PROBE

DETECT PROBE'S SIGNAL

FIG. 1. Basic steps in a DNA probe hybridization assay. The sample containing cells is captured (or blotted) on a solid support such asa nitrocellulose filter disk. The cells are then lysed to release the DNA, which binds to the solid support. Usually the filter is washed, andunreacted sites on the filter are blocked. A labeled probe is then added and allowed to hybridize to the sample DNA. Unbound probe iswashed away, and the hybridized probe is detected.

to Sepharose beads, has also been used to separate DNAfrom solutions (5). After the DNA has been chemicallyreleased from the sample, the methidium binds the DNA tothe Sepharose beads, and contaminants can be removed bywashing. The DNA is then released from the dye with adilute base and collected.A homogeneous hybridization assay format that recently

became commercially available is based on the protection ofa phenol ester of an acridinium ester chemiluminescentprobe label (1, 71). In this assay, if the probe is hybridized tothe target nucleic acid, the phenyl ester is protected fromhydrolysis. However, unhybridized probe does not protectthe acridinium ester from hydrolysis, resulting in loss ofchemiluminescence when the unhybridized probe label is

released. The hybridization protection assay format permitsthe use of large concentrations of probe without backgroundproblems (1). The resulting hybridization reactions are com-plete within 10 min and have a sensitivity of 10-16 to 10-21mol of nucleic acid (1).

AMPLIFICATION METHODS

Amplification of very minute quantities of nucleic acidsbefore hybridization assays are performed holds great prom-ise for successful commercialization of DNA probes for usein clinical and anatomical laboratories. Most infectiousagents or cellular materials of interest are available only inlimited quantities unless they are first cultured. Some infec-

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TARGET DNA

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IS PRESENTFIG. 2. Solid-phase sandwich hybridization assay. The target

nucleic acid acts as a bridge joining the bound capture probe with afree probe that has the label attached. After the mixture has beenwashed, signal probe detection indicates the presence of the targetnucleic acid. Modified and reprinted from the Joumal of FoodProtection (103a) with permission of the publisher.

tious agents cannot be cultured, and others often require along period of culture before hybridization assays will detectthem. To perceive limited amounts of nucleic acids withDNA probes without cell cultivation, some method of en-hancing the detection is required. With the rapidly emergingmethods of amplifying nucleic acids, the future use of DNAprobes in clinical and anatomical laboratories is almostassured.Probe assays can be amplified by either of two general

strategies or a combination of both. The target nucleic acidsequence can be amplified, thus increasing the amount oftarget nucleic acid available for detection. Alternatively, theprobe can be equipped with an amplification system that willallow a very small hybridization reaction to generate adetectable signal.

Polymerase Chain Reaction

In 1983, a conceptually simple process of amplifying, or

increasing, the number of specific nucleic acid fragmentspresent in a sample was developed by Mullis (70). In 1989,this process was deemed the "major scientific development"of the year (34). This process is termed polymerase chainreaction (PCR). In just a few years and after some refine-ments, PCR has become the most frequently used methodfor amplifying nucleic acids, especially DNA. The PCRtechnique is based on the reiteration of a three-step process(Fig. 5): denaturing dsDNA into single strands, annealingprimers (specific synthetic oligonucleotides) to the ssDNA,

HYBRIDIZATION OF TARGET TO

CAPTURE PROBE DISPLACES

SIGNAL PROBE (WEAKLY BOUND)

------ .FREE SIGNAL PROBE

FIG. 3. Strand displacement hybridization assay. A captureprobe has a weakly hybridized signal probe attached. Addition ofthe target nucleic acid displaces the more weakly bound signalprobe. Detection of the signal in solution or absence of the signalprobe from the solid support after washing indicates that hybridiza-tion of the target nucleic acid has occurred. Modified and reprintedfrom the Journal of Food Protection (103a) with permission of thepublisher.

and enzymatically extending the primers complementary tothe ssDNA templates. After the primers are annealed to thedenatured DNA, the ssDNA segment becomes the templatefor the extension reaction. Nucleotides, present in the solu-tion in excess, are enzymatically joined to form the cDNAsequences. During the second and subsequent cycles, theoriginal DNA segment and the newly generated cDNAstrands become templates. Each cycle of PCR thereforedoubles the amount of specific DNA present. A ty picalamplification is 20 to 40 cycles and results in a 10'-foldamplification of the original DNA (77, 94). The improvementof PCR by the use of a thermostable enzyme, Taq DNApolymerase, has allowed semiautomation and simplificationof the process to the point that it has become widely adoptedin research laboratories (26, 40).PCR does have some shortcomings. The system is suscep-

tible to contamination with extraneous DNA fragments thatcould be amplified along with the sample. This is importantin that extraneous DNA can be carried over from previousamplifications (amplicons) or introduced from other sources.

To eliminate this problem, adherence to careful laboratoryprocedures, such as rigid quality control of enzyme prepa-rations and the use of dedicated pipettes and prealiquotedreagents, is necessary. Various other techniques for reduc-ing extraneous DNA contamination of PCR products havebeen described (17, 21, 22, 47, 54, 75, 79, 82, 83, 85).

5' DNA 15'TARGET DNA 3'

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FIG. 4. Dipstick hybridization assay. A variation on sandwich hybridization, the dipstick hybridization assay can be used for either DNAor RNA targets. By extensively washing the solid phase and changing reaction containers, nonspecific background interference is reduced.Abbreviation: HRP, horseradish peroxidase. Modified and reprinted from the Journal of Food Protection (103a) with permission of thepublisher.

Methods to eliminate or modify the amplicons so that theydo not contaminate subsequent PCR amplifications (steril-ization) are also being developed (27, 78). In one method(18), the sterilization process is the result of treating the PCRamplicons with 4'-aminomethyl-4,5'-dimethylisopsoralen(4'-AMDMIP), an isopsoralen derivative that reacts withpyrimidine to form a cyclobutane ring when photoactivatedwith 320- to 400-nm light. The cross-links formed effectivelyblock the polymerase extension reactions. However, theprocess must be optimized for each reaction on the basis ofthe length and sequence of the amplicon and on the amountof carryover tolerable. Another method uses the enzymeuracil DNA glycosylate (62). Uracil DNA glycosylate re-moves the uracil base from the sugar-phosphate backbone ofssDNA or dsDNA. The resulting apyrimidinic site cannot bereplicated by polymerase. The result is that either a DNA

primer that was synthesized with uracil in place of thymidineor an amplicon that was generated with uracil in place ofthymidine can be sterilized by the addition of uracil DNAglycosylate. Normal PCR temperature processing inacti-vates uracil DNA glycosylate. One of the simplest methodsfor sterilization of PCR reactions is the use of UV radiation(75, 83). Use of UV light to dimerize extraneous or leftoverDNA, together with a dedicated work area and appropriatePCR handling techniques (54), normally reduces the contam-inating DNA to a level that can be tolerated. Cimino et al.(17), however, recommend caution in relying on UV radia-tion to completely solve the contamination problem.PCR requires much technical skill and some special labo-

ratory equipment. Although PCR has been semiautomated,the technique still requires a significant amount of labor forsample preparation and subsequent detection of DNA. The

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TARGET FORAMPLIFICATION

DENATURE TARGET

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FIG. 5. PCR amplification. PCR amplification involves cycles of denaturing, reannealing (hybridizing), and enzymatically addingnucleotides. In the first step, DNA is denatured. The ssDNA is then allowed to reanneal so that most single strands of DNA hybridize toprimers that were added to the mixture. The primers serve as the starting points for the enzymatic addition of nucleotides, with the originalDNA strands serving as templates for the new DNA being made. After the first cycle, twice as many DNA sequences exist; these serve astemplates for the synthesis of more DNA fragments during the next cycle. Amplification normally consists of 20 to 40 cycles. Different cyclingtimes and temperatures can be used, but most cycles are in the range of minutes, so a complete amplification procedure can be completedwithin a few hours. Modified and reprinted from the Joumal ofFood Protection (103a) with permission of the publisher.

protocols are not yet sufficiently developed and reproduciblefor many assays (27, 78), but improvements in PCR technol-ogy will undoubtedly be made. There are currently manyvariations of PCR, including PCR using nested primers,inverse PCR, anchored PCR, and amplification refractorymutation system (6, 72). PCR technology and methods havebeen reviewed by Erlich et al. (27), Persing (78), and Bej etal. (6).

Ligase Amplification ReactionThe ligase amplification reaction (104), also called the

ligase chain reaction (LCR is the trade name) (4, 56, 94, 103)or the oligonucleotide ligation assay (27, 73), is anothertechnique for detecting or amplifying a target sequence.Unlike PCR (which creates new DNA molecules from indi-vidual nucleotides), the ligase reaction uses a thermostable

enzyme (2, 4) to join two oligonucleotides that are immedi-ately adjacent to each other (Fig. 6). Like PCR, this reactionis cyclic; denaturation, annealing, and ligation are the basisfor the amplification reaction. As in PCR, the ligated oligo-nucleotide pairs, along with the original sequence, becometemplates for the next cycle (104). From 20 to 30 cycles ofthis reaction yield a 106-fold increase in the original target.The method is currently able to detect as few as 10 nucleicacid targets (2).

In the ligase reaction, the entire target sequence must beknown, because a 1-bp mismatch at the point of ligation canprevent ligation of the oligonucleotides. Although this couldbe detrimental in many situations, failure to ligate can beused to detect point mutations in the target sequence.Barany used this approach to differentiate normal and sicklep-globulins in blood samples from patients with sickle cell

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3-~~~~~~~~~~55U1 11111111153, TARGET DNA

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FIG. 6. Ligase amplification of a target by use of two oligonucleotide probes. Two probes that hybridize adjacent to each other on a targetstrand are obtained and ligated with T4 DNA ligase into a longer sequence. The ligated probes can also function as a template for furtherhybridizations and thereby amplify the original target DNA in a cyclic reaction system. The ligase reaction has also been used to ligate twoprobes in a solid-phase extraction system to produce cleaner reactions. Modified and reprinted from the Journal ofFood Protection (103a)with permission of the publisher.

anemia (4). As with PCR, a thermostable ligase is necessaryfor the cyclic amplification reaction. However, the ligasereaction appears better suited than PCR for diagnostic work.Whereas PCR generates new DNA or RNA molecules fromnucleotides, in many situations the newly generated mole-cules may still require some form of verification or identifi-cation to determine the exact specificity of the DNA. Theligase reaction makes use of known sequences for the solepurpose of detection; the probes must hybridize to adjacentsites in order to be ligated together and detected.Bond et al. described the use of LCR to detect human

papillomavirus (10). In their approach, both a separationstep and a signal detection mechanism are incorporated intothe oligonucleotide probe.Another variety of ligase reaction involves the use of both

a polymerase and a ligase (8). Two oligonucleotide primersare annealed to the target but are spaced so that polymerasefills a gap between the two primers; the ligase then connectsthe filled gap and the second primer. This modification,called gapped-LCR (G-LCR is the trade name), has a Euro-pean patent pending.

In addition to Barany's detection of the mutation that

causes sickle cell hemoglobin (4), the ligase amplificationreaction has been used to diagnose the mutation responsiblefor cystic fibrosis (73), for detection of gene segments in thehuman T-cell receptor 1-chain locus (73), and for detectionof human papillomavirus (10).The final development and marketing of ligase reactions

may depend on rulings by the U.S. Patent Office and thecourts on conflicts between various patent claims (102).

Transcription-Based Amplification Systems

In 1989, Kwoh et al. (53) described another technique fornucleic acid amplification, a transcription-based amplifica-tion system (TAS). TAS is a two-cycle scheme based onDNA synthesis and RNA transcription. In this procedure,denatured DNA or a strand of RNA is the target for anoligonucleotide primer that contains a polymerase-bindingsite. After hybridization of the primer to the target, a reversetranscriptase elongates the primer complementaxy to thetarget sequence. After heat denaturation, another oligonu-cleotide primer is annealed to the new DNA strand. Reversetranscriptase is again added to produce a dsDNA molecule.

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PolymeraseElongation I

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- || DNA Assay ||

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Polymerase 1;ILPolymerase PolymeraseElongation I~ ~ P9I JrerseElongation

DNA Assay | AssayZ|UFIG. 7. 3SR amplification. 3SR amplification is based on the use of probe primers that have polymerase-binding sites (grey boxed areas)

for the action of reverse transcriptase. The reverse transcriptase and RNA polymerase work together to amplify a DNA or RNA targetwithout thermal cycling. The arrows indicate possible uses of the DNA sequences created: recycling for further amplification or use in a DNAassay.

By the addition of an RNA polymerase, multiple copies (10to 1,000 per cycle) of RNA are produced. In this method,only four cycles of the reaction are necessary to obtain up to106 copies.

Self-sustained sequence replication (3SR is the tradename) RNA transcription is a modification of TAS (31, 33).The 3SR reaction differs from the TAS in that 3SR isisothermal (37 to 42°C) and the RNA target is degraded (Fig.7). The procedure also can be used to amplify DNA if aninitial denaturing step is included. RNase H is used todegrade the RNA-DNA duplexes formed in the TAS reac-tion and allows for conversion to a dsDNA that has apolymerase-binding site on each end. The dsDNA continuesthe cycle by acting as template for the synthesis ofRNA thatreenters the reaction. A 105-copy amplification occurs within15 min; it would take PCR 85 min to amplify that much if theprocedure were 100% efficient.

The advantages of 3SR over PCR are that no thermalcycling is required. Also, since the 3SR reaction is based inpart on the transcription of RNA, multiple copies of RNAare transcribed in each cycle. This means that fewer cyclesare needed for 3SR to generate the same amount of nucleicacid that PCR generates. In addition, 3SR can distinguishDNA and RNA targets. Like the other amplification meth-ods, 3SR is subject to contamination by extraneous nucleicacids. It is also suffers from a low inherent specificitybecause the stringencies of the reactants are lower than withother amplification methods and because multiple enzymesare used.

Nucleic acid sequence-based amplification (NASBA is thetrade name) is another commercial development of theisothermal TAS method (45, 63) and is very similar to 3SR(20, 94). In 1989, a European patent on NASBA was filed(94).

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DenatureTaraet

I

5'""""|| 5

Hybridize 31DNA-RNA-DNA I

Probe_"P......... b~~~~~~~~~~~~41

51 se~~~~~~~~5

1 ~~~~~3,

Detect ProbeFragments

/RNAse H Fragments

Dissociate

FIG. 8. Cycling probe reaction. The cycling probe reaction uses RNase H to degrade the RNA in an RNA-DNA hybrid probe. The probefragments then dissociate from the target and can either accumulate to a detectable level or be treated further (amplified or modified).

Six cycles of TAS have been shown to be capable ofdetecting one human immunodefiency virus (HIV)-infectedlymphocyte in a field of 106 uninfected cells (30). Nucleicacid amplification with 3SR has been used to analyze andmonitor 3'-azido-3'-deoxythymidine (zidovudine) drug resis-tance in HIV (29). Compton (20) reported that NASBAdetected "as few as three HIV particles" in a 100-,ul sampleof human plasma, but a later report by Kievits et al. (45)reported that the limit of detection was 10 HIV particles orfive infected cells.

Cycling Probe Reaction

The cycling probe reaction is almost the exact opposite ofLCR. In this procedure, a nucleic acid probe that incorpo-rates DNA-RNA-DNA sequences is synthesized (Fig. 8).After the probe is allowed to hybridize to the target, RNaseH, an enzyme that specifically degrades the middle RNAportion of the probe, is added. The DNA fragments thatremain dissociate from the target. The target is now free tohybridize with another probe molecule; therefore, the reac-tion is inherently cyclic without external manipulations. Thefree DNA fragments can then be detected or amplified by asecondary amplification system (23). This is a relatively newmethod, and further development is needed before it can becommercialized.A similar amplification method is based on the use of

restriction enzymes to cleave an oligonucleotide probe. Inthis technique a labeled oligonucleotide and a restrictionenzyme are used to cut the labeled probe into a shorterproduct. With the use of a second complementary oligonu-cleotide probe, the reaction can become isothermally cyclic.

Q3 Replicase AmplificationAnother method of nucleic acid amplification that is being

commercially developed is known as Q,B replicase amplifi-

cation (16, 52, 59, 94, 99). This amplification method isnamed after the major enzyme responsible for the amplifi-cation, the replicase of the Qpi bacteriophage. The enzyme isan RNA-directed RNA polymerase, meaning that it enzy-matically assembles RNA from an RNA template. The Q,Benzyme comprises four subunits, of which one is from theQI bacteriophage and three are from the phage host E. coli(14, 60). This amplification system was first reported byHaruna and Spiegelman in 1965 (36). Later, an RNA mole-cule called midivariant 1 (MDV-1) was isolated. MDV-1 actsas the specific target for the Q3 replicase and allows thereplicase to be used in nucleic acid amplifications (42).Following sequencing of the MDV-1 molecule (66), it waslearned that a short nucleic acid sequence could be insertedinto one of the complementary loops within the MDV-1molecule without harming replication of the molecule (65).This modified MDV-1 could then be used as a hybridizationprobe (65).Although both PCR and Qp replicase amplify nucleic

acids, the amplification of RNA serves as a reporter systemfor signal detection rather than as a method of increasing theamount of target nucleic acid as in PCR (Fig. 9). Theincrease in the amount of RNA can readily be detected byusing an intercalating fluorescent dye such as ethidiumbromide. In less than 15 min, Q1 replicase can amplify anMDV-1 RNA molecule into 106 to 109 RNA molecules (14,48). One thousand RNA molecules can be amplified to 125 to200 ng, a 109-fold amplification! Because of the synthesis ofmultitudinous RNAs, hybridization reactions can be mea-sured by simple colorimetric techniques (59). Quantitation ofthe reaction is possible because of the kinetics, which allowconstruction of a standard curve and determination of theinitial concentration of bound complex (99).Another innovation in the use of Q3 replicase amplifica-

tion came with the development of reversible target capture(14, 68). A poly(dA)-tailed capture probe and magnetic

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5'111111111111111111* so5'3'

TARGET DNA

IT

III IliIIII I" 7r"*51SU"''"'''"'''''~~31

DENATURED TARGET

ITCAPTURE PROBE

MDV-1 WITHINSERTED PROBE

ALLOW MIXTURE TO HYBRIDIZE AND WASH

AWAY UNHYBRIDIZED MDV-1 PROBE

_%, _- I

' d

AMPLIFICATION WITH Q-B REPLICASE (TRANSCRIPTION)YIELDS A DETECTABLE RNA INCREASE

FIG. 9. Q1 replicase amplification. Q1 replicase amplifies the presence of a target by the transcription of copious amounts of RNA.Modified and reprinted from the Joumal ofFood Protection (103a) with permission of the publisher.

microparticle beads containing poly(dT) are used to wash thereaction mixture clean of any residual contaminating mate-rials, thereby lowering background noise (14, 59, 61). Theuse of chaotropic salts also aids Q1 replicase amplification.The salts lyse cells, denature proteins (including nucleases),and liberate and linearize DNA without interfering in hybrid-ization of the MDV-1 probe (61, 90).

Modifications to the use of MDV-1 RNA as a probeinclude adding a "molecular switch" to a region of the probeor using probes that are not replicable but can synthesizereplicable reporter RNA (51, 60). A molecular switch couldbe used to help clean the reaction system and eliminateunbound probes that might cause a false-positive reaction.For example, the probe sequence could be incorporated intothe RNase III-binding site on the MDV-1 molecule (Fig. 10).If the MDV-1 molecule is not hybridized, the RNase-bindingsite is intact and available for RNase destruction. If theMDV-1 molecule is hybridized, the probe sequence obliter-ates the complementary loop structure that forms the RNaseIII binding site. MDV-1 RNA can also be synthesizedthrough the action of two separate probes that each containa polymerase promoter sequence and a sequence that canserve as a primer for its own extension. In two separate

hybridizations and extensions, a dsDNA promoter regionthat can, with the aid of a polymerase, transcribe a replicat-able RNA is made. The replicatable RNA can then beamplified by QpI replicase.Although the tests are not commercially available, one

company has used the Q,l replicase nucleic acid amplifica-tion to detect both HIV and cytomegalovirus (57). In detec-tion of synthetic HIV target RNA, 600 molecules could bedetected in approximately 2.5 h (57).

Strand Displacement Amplification

Another isothermal amplification method is based on thedisplacement of one probe when DNA polymerase is used toextend a second probe. In the original method (98), the targetDNA underwent initial cleavage into 47-bp fragments andheat denaturation. A probe containing a HincII recognitionsite on the 5' end was hybridized to the target fragments.When it is bound to the target site, the probe possessing theHincII recognition site (6 nucleotides) and several morebases that do not hybridize to the target nucleic acid isextended past the end of the target fragments (Fig. 11). DNApolymerase then generates a nucleic acid strand complemen-

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HYBRIDIZE MDV-1 AND TARGET

/ MDV-1WITHPROBEINSERT

51 M IIIIIIIIIIII « 5 TARGET DNA

Denature Target DNAand Hybridize Four.....%/%5 Probes

3

Extend Probes andDisplace Down Stream

____t _ Probe

Displaced Probe, Becomes Target for the

,'-\OtOther Set of Probes

/14

SEQUENCE

NONFUNCTIONALRIBONUCLEASEBINDING SITE

PROBE -ISEQUENCE

FUNCTIONALRIBONUCLEASEBINDING SITE

FIG. 10. Molecular switch. RNase III is an enzyme that destroysdsRNA. Incorporation of the probe sequence into the end of acomplementary loop structure will cause the loop structure tobecome linear on hybridization. Loss of the RNase-binding site (thecomplementary double-stranded region) on hybridization will makethe probe resistant to destruction. Probe sequences that are notbound become susceptible to destruction and prevent nonspecificsignal generation.

tary to the target and free end of the probe, incorporating amodified dATP (dATPaS). The HincII enzyme then nicksthe probe strand at the HincII recognition site (the hemi-phosphorothioate HinclI recognition site prevents completecleavage of the dsDNA). DNA polymerase then generates anucleic acid strand that is complementary to the targetnucleic acid strand and that displaces the original probesegment from the target fragment. Because of the orientationof the HincII recognition site, the complementary probesequence generated by DNA polymerase retains a functionalHincII recognition site. This allows continual nicking of theprobe by HincII and subsequent displacement through theaction of DNA polymerase.To partially overcome background amplification of non-

target DNA, the method was modified to eliminate initialtarget cleavage (97). Using four probes, the reaction pro-ceeds as shown in Fig. 11. The simultaneous extension of allfour primers displaces two probes that can act as targets forthe opposite probes. The amplification then proceeds withthe nicking of the probe HincIl recognition site and displace-ment by the original method. The resulting action of DNApolymerase on the probes amplifies nucleic acid exponen-tially.

Strand displacement amplification has been used to am-plify Mycobacterium tuberculosis genomic DNA to 107copies in 2 h (97). Like PCR, certain experimental parame-ters impose some limitations (97) on the procedure.

I Extend Probes andDisplace

Downstream Probe

4, EnzymaticallyNick Probe

I Extend DNA andDisplace Probe

Displaced Strands Become Targetsand dsDNA Reenters Cycle

FIG. 11. Strand displacement amplification. Strand displacementamplification is based on the displacement of an enzymaticallyextended DNA probe by the extension of another enzymaticallyextended DNA probe located upstream (5' to the first probe). Withthe incorporation of a restriction endonuclease site that can benicked, creating a second probe system, the reaction becomes cyclicwithout the need for temperature changes after the initial denatur-ation step. Two probes hybridize each strand of the target sequencein the initial step. Both probes are enzymatically extended at thesame time, with the upstream probe displacing the downstreamprobe. The displaced probe becomes the target for the oppositestrand's probe. The restriction site is oriented in such a way that thedsDNA is only nicked instead of cleaved. This nick allows the nicksite to act as an extension site for polymerization of cDNA thatdisplaces the downstream DNA. The displaced DNA becomesanother target sequence. The creation of displaced strands generatesthe amplification effects of the method.

SIGNAL AMPLIFICATION AND DETECTIONMETHODS

Amplification of the detectable signal also improves thesensitivity of nucleic acid probes. A properly constructedprobe system that hybridizes even one target can be detectedif the signal is amplified sufficiently. Current probe assayscannot detect a single hybridization reaction, but the follow-ing methods increase the sensitivity of signal detection and,in some instances, provide very substantial signal amplifica-tion.

Several nonisotopic test detection systems that are being

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developed are based on chemiluminescence. One systemuses acridinium ester-labeled probes. The reaction is initi-ated by the addition of hydrogen peroxide (77, 87). Light isproduced by decay of the ester as a result of the effect ofhydrogen peroxide, and the amount of light is proportional tothe amount of target nucleic acid present. This reaction iscompleted within seconds; however, a chemiluminometer isrequired to detect the small quantities of light emitted (77).Another chemiluminescent compound is phenylphosphate-substituted dioxetane (84). This compound undergoes con-version to a luminescent form in the presence of alkalinephosphatase; it is reportedly 10 times more sensitive thancolorimetric detection with o-phenylenediamine or horse-radish peroxidase (19). Chlorine- and bromine-substituteddioxetanes have also been described (12); these improvechemiluminescent detection by eliminating nonenzymati-cally activated chemiluminescence, and they have shortenzyme activation times. Another approach being developedis the use of a photochemical sensitizer as a label (64).Oxygen transfer of light excitation molecules to olefin resultsin generation of a dioxetan that, when heated, emits light. By

using filters, background noise can be reduced; this in-creases the sensitivity of the assay.The generation of a chemiluminescent reaction at the

surface of an electrode by means of an electrical andchemical excitation is termed electrochemiluminescence.This process provides another rapid (<25 min) and sensitive(200 fmol/iter) detection method for nucleic acids (9, 43).The commercially available label uses a Tris-rutheniumbipyridyl complex (9, 43). The Tris-ruthenium bipyridylcomplex and a reductant such as tripropylamine undergooxidation-reduction at the electrode surface, and a photon(detected at 620 nm) is emitted (9). The Tris-rutheniumbipyridyl complex can be regenerated to form the basis of acyclic type of reaction that can amplify the signal (9).Instrumentation for detecting this reaction is reported to besimple, and the process can be automated (9).Another nonisotopic method involves the use of enzyme-

labeled antibodies. Labeled antibodies are targeted against acompound that is covalently linked to or incorporated intothe probe. For example, probes with modified cytotidineresidues are labeled with fluorescein. High-affinity antifluo-

5'111111111111111111119 5'31

TARGET DNA

ENZYME LABELED PROBE

FIG. 12. Branched DNA signal amplification. Signal amplification, based on the use of branched DNA oligonucleotides, involves targetcell lysis and denaturation of the target nucleic acid, addition of the capture and label probes, solid-phase extraction and washing, additionof the branched DNA multimer, another washing step, addition and hybridization of an enzyme-labeled probe, and detection of the enzymaticsignal.

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NUCLEIC ACID-BASED DETECI1ON METHODS 383

31

5' 1111111111 5'

TARGET DNA

1T,.

DENATURE TARGET

SIGNAL PROBE 1

REACTANTS \

SIGNAL PROBE 2

each piece of the probe is labeled with one component of atwo-component interacting fluorophore or enzyme system(15, 95). When the two separate probes hybridize besideeach other, excited energy of the fluorophore is transferredto the now-adjacent acceptor fluorophore or enzyme system.The acceptor is activated, and a signal is created. A separa-tion step is not required because without hybridization, thetwo components are not close enough to each other togenerate a signal.

NO SIGNAL GENERATED WHENREACTANTS ARE NOT CLOSE

\ /SIGNAL PROBES AND DNA

ARE ALLOWED TO HYBRIDIZEBRINGING REACTANTS TOGETHER

315'

SIGNAL EMITTEDFIG. 13. Signal generation assay. This involves signal amplifica-

tion through the use of two separate probes that, when hybridizedclose together, generate (or change) a signal that is easily detected.If the probes are not hybridized close to each other, their reactivecomponents cannot react; in solution, they are kept far apart bynormal solution forces. Modified and reprinted from the Journal ofFood Protection (103a) with permission of the publisher.

rescein antibodies are labeled with horseradish peroxidaseand used to detect the probe (77). Instead of fluorescein,sulfonation of cytosine, introduction of 2-acetylaminofluo-rene groups into guanine and its derivatives, and incorpora-tion of digoxigen-labeled dUTP into DNA have been usedwith enzyme- or fluorophore-conjugated antibodies (24, 44,77, 89, 99).A nucleic acid-based method of amplifying the detection

signal involves the use of branched oligonucleotides (91, 93).Branched oligonucleotides that have a primary sequenceconceived to directly or indirectly hybridize to a targetnucleic acid have been synthesized (only the indirect methodis shown in Fig. 12). The secondary sequences of thesynthesized oligonucleotide serve as hybridization sites foran enzyme-labeled oligonucleotide. The primary and sec-ondary sequences are covalently linked through branchpoints (39). Signal amplification results from the attachmentof 60 to 300 enzyme molecules (92) to each nucleic acidtarget (Fig. 12). The method is dependent on solid-phaseseparation and washing to reduce background interferencebefore the addition of the branched oligonucleotide. Whenthis method was combined with the sensitivity of chemilu-minescence, as few as 1,000 viral hepatitis genomes weredetected directly in human serum or plasma (91). Themethod has also been applied to the detection of Chlamydiatrachomatis, Neisseria gonorrhoeae, and penicillin and tet-racycline resistance plasmids (92).Although not a true signal amplification, probe-based

signal generation is a unique method of generating a signal onthe basis of nucleic acid hybridization. In probe-based signalgeneration (Fig. 13), a single-stranded probe is cut in half and

AUTOMATION

Several authors (3, 9, 38, 60, 64, 73) include the subject ofautomation in their discussions of the use of probes. Hybrid-ization reactions definitely have the potential to be auto-mated. At this time, except for one automated slide stainerthat can be set up for in situ DNA hybridization, nocompletely automated systems are commercially available.Until automated systems become available, the use of rou-tine nucleic acid amplification-detection methods in clinicallaboratories outside of the academic or major medical centerwill be limited.One method that holds promise for automation of DNA-

based detection methods is based on a class of sensors calledbiosensors. A biosensor is an electronic-based device thatgenerates a signal, either electrical or optical, when biolog-ical molecules bind to a sensor. These biological moleculescan be carbohydrates, nucleic acids, or proteins such asenzymes, hormones, antigens, and antibodies. One applica-tion, for DNA hybridization detection, is based on surfaceplasmon resonance (86). This method is based on reductionin the intensity of reflected light and changes in refractiveindex that occur during hybridization of DNA. The system issensitive enough to detect DNA hybridization on the surfaceof a sensor that is being illuminated with laser light andscanned with photodiode detectors. The system can detect320 fg (3.2 x 10-13 g) of a 97-bp molecule or 24 fg of a7,200-bp DNA molecule (compared with 100 fg of DNA on aSouthern blot).Analogies between immunoassay development and prog-

ress in nucleic acid detection methods are appropriate. Thefirst immunoassays were research laboratory methods thatwere subsequently transferred to clinical laboratories. Still,immunoassay automation probably has not peaked. Theultimate conception of an automated DNA probe analyzerwould be like a "black box" that could be loaded with aclinical specimen and started and that would, in less than 1 h,indicate that each of multiple target organisms was or wasnot present. Whether or not this black box evolves asLizardi and Kramer's conception of a multiplex diagnosticassay (60) with a membrane strip that includes an arrange-ment of defined DNAs that bind amplified products, thefuture of automation of DNA probe methods holds muchexciting promise.

SUMMARY AND CONCLUSIONS

Tenover (89) summarized the use of DNA probes asfollows: "The goal of DNA probe technology is to eliminateroutine cultures, whether they be bacterial, viral, or fungal."This is a very ambitious statement but one that mightsomeday be fulfilled if the technology continues to progressas it has. Currently, these methods are more suitable forresearch laboratories than for routine clinical laboratories.As patent disputes are resolved and the processes becomemore refined, automated, and standardized, nucleic acid

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detection methods will displace or supplement current meth-ods of rapid clinical laboratory diagnosis.

ACKNOWLEDGMENTS

Support in the form of a Graduate Research Assistantship wasprovided by a U.S. Department of Agriculture grant to the FoodSafety Consortium.The encouragement and comments of Paul A. Hartman are

appreciated.

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