Michael C. Little et al- Strand Displacement Amplification and Homogeneous Real-Time Detection Incorporated in a Second-Generation DNA Probe System, BDProbeTecET

8/3/2019 Michael C. Little et al- Strand Displacement Amplification and Homogeneous Real-Time Detection Incorporated in a Second-Generation DNA Probe System, B

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Strand Displacement Amplification andHomogeneous Real-Time Detection Incorporatedin a Second-Generation DNA Probe System,

BDProbeTecETMichael C. Little,* Jeffrey Andrews, Richard Moore, Silvia Bustos, Lynda Jones,Chris Embres, Gerard Durmowicz, James Harris, Dolores Berger, Karen Yanson,

Christine Rostkowski, Daretta Yursis, James Price, Thomas Fort, Adriann Walters,Matthew Collis, Oscar Llorin, Janet Wood, Frank Failing, Christian OKeefe,

Brian Scrivens, Bill Pope, Tim Hansen, Ken Marino, Keith Williams, andMichael Boenisch

Background: Amplified DNA probes provide powerfultools for the detection of infectious diseases, cancer, andgenetic diseases. Commercially available amplificationsystems suffer from low throughput and require decon-tamination schemes, significant hands-on time, and spe-cially trained laboratory staff. Our objective was todevelop a DNA probe system to overcome these limita-tions.Methods: We developed a DNA probe system, theBDProbeTecTMET, based on simultaneous strand dis-placement amplification and real-time fluorescence de-tection. The system uses sealed microwells to minimizethe release of amplicons to the environment. To avoidthe need for specially trained labor, the system uses asimple workflow with predispensed reagent devices; aprogrammable, expandable-spacing pipettor; and the96-microwell format. Amplification and detection timewas 1 h, with potential throughput up to 564 patientresults per shift. We tested 122 total patient specimensobtained from a family practice clinic with the BDProbeTecET and the Abbott LCx amplified system forthe detection of Chlamydia trachomatis and Neisseria

gonorrhoeae.Results: Based on reportable results, the BDProbeTecETresults for both organisms were 100% sensitive and100% specific relative to the LCx.

Conclusions: The BDProbeTecET is an easy-to-use,high-throughput, closed amplification system for thedetection of nucleic acid from C. trachomatis and N.gonorrhoeae and other organisms. 1999 American Association for Clinical Chemistry

The amplified detection of nucleic acids has beenachieved using a variety of techniques, including stranddisplacement amplification (SDA),1 PCR, ligase chainreaction, nucleic acid sequence-based amplification, andtranscription-mediated amplification (15). Each of theseamplification methods uses different approaches toachieve the amplification of low copies of nucleic acid toamounts that can subsequently be detected. Detectionmethods are typically performed after the amplificationstep and include such approaches as colorimetric detec-tion, chemiluminescence, and gel electrophoresis detec-tion (6, 7 ). The shortcomings of these systems include lowthroughput, significant hands-on time, complex work-flow, and the potential for amplicon contamination. Theseshortcomings have generally limited the adoption ofamplified probe methods for routine use. Furthermore, to

avoid false-positive results, the generation of ampliconsduring the amplification process requires separate ampli-fication and detection areas, and sometimes multiplerooms or chemical or enzymatic schemes to reduce theirconcentrations (8, 9 ). Although semi-automated systems

Becton Dickinson Microbiology Systems, 54 Loveton Circle, Sparks, MD

21152.*Author for correspondence. Fax 410-316-3690; e-mail Michael_C_Little@

ms.bd.com.

Received January 21, 1999; accepted March 25, 1999.

1 Nonstandard abbreviations: SDA, strand displacement amplification; ET,energy transfer; CT, Chlamydia trachomatis; GC, Neisseria gonorrhoeae; EB,

elementary body; and AC, amplification control.

Clinical Chemistry 45:6777784 (1999) Molecular Diagnostics

and Genetics

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have been introduced, throughput, hands-on time, andother shortcomings still exist.

For amplified DNA probe technology to become moreroutine and to be adopted into clinical laboratory settingshaving fewer skilled technologists, there is a need forsimpler, higher throughput and more user-friendly sys-tems. Recently, methods using real-time detection of

amplified nucleic acids have been described (5, 10, 11 ).These methods represent the future in molecular diagnos-tics and may soon allow laboratories to attain these goals.However, none of these methods has been adapted intocommercially available user-friendly systems for diagnos-tic settings.

We describe the first of these second-generation sys-tems, the BDProbeTecET. This system is based on thesimultaneous amplification of nucleic acids by SDA andreal-time detection using fluorescence energy transfer(ET). When the BDProbeTecET system is applied to thedetection of Chlamydia trachomatis (CT) or Neisseria gonor-rhoeae (GC), as few as 1015 GC cells or CT elementary

bodies (EBs) can be detected reliably in 1 h on theinstrument. The system configuration and workflow per-mits a throughput of up to 564 patient results per shift.

Materials and Methods

assay consumables and buffersThe consumables used in the assay include two sets ofmicrowells. The microwells are provided as strips ofeight, which can be broken into individual tests if needed.The microwells are color-coded on the basis of assay type.For each test, two microwells are needed. One microwell(Priming Microwell) contains dried SDA primers, one ofthe four nucleotides, and fluorescent oligonucleotideprobe. A second microwell (Amplification Microwell)contains the remaining dried SDA reagents, includingSDA enzymes. Processed specimen is used to rehydratethese reagents. All CT and GC microwells and samplediluents were provided by Becton Dickinson Microbiol-ogy Systems (Sparks, MD).

target regions, primers, and probesSDA has been previously described (15), and its princi-ple is summarized in Figs. 1 and 2, which are discussedfurther under Results. Detection utilizes fluorescence ETas described in Fig. 3A and as discussed with the dataunder Results. For the amplification and detection of CT,the multicopy cryptic plasmid (12) was chosen as a target

region. The region being amplified spans the followingsequence: 5-CAGCAAATAATCCTTGGGACAAAATC-AACACCTGTCGCAGCCAAAATGACAGCTTCTGATG-GAATATCTTTAACAGTCTCCAATAATTCATCAACCA-ATG-3. The SDA amplification primer and bumperprimer pairs (target-binding region underlined, BsoBIrestriction site bold and italicized) are as follows: 5-ACCGCA TCG AAT GCA TGT CTC GGG GAG ACT GT-TAAA GAT A-3 and 5-CAT TGG TTG ATG AAT TATT-3; 5-CGA TTC CGCTCC AGA CTT CTC GGG ACA

AAA TCA ACA CCT G-3 and 5-CAG CAA ATA ATCCTT GG-3. The detector probe utilized for real-timedetection is 5-(Fam)-TAG CAC CCG AG TGCT (Rox)-CGC AGC CAA AAT GAC AGC TTC TGA TGG AA-3.

For the amplification and detection of GC, a regionwithin the multicopy pilin gene-inverting protein homo-logue (13) was chosen. The target region spans the

following sequence: 5-CGCAAATCATCAAAGCCAT-GAATGAACAGCTTGAAGTTTTAAAGGAGAAGATA-AAAGAGCAGACGGAGAAGCCTAACTGCAAGGAA-GGCGTGAAGCGTCTTGA-3. The SDA amplificationprimer and bumper primer pairs (target-binding regionunderlined, BsoBI restriction site bold and italicized) areas follows: 5-CGA TTC CGC TCC AGA CTT CTC GGGAAC AGC TTG AAG TTT T-3 and 5-CGC AAA TCATCA AAG-3; 5-ACC GCA TCG AAT GCA TGT CTCGGG TCC TTG CAG TTA GGC-3 and 5-TCA AGA CGCTTC ACG-3. The detector probe utilized for real-timedetection is 5-(Fam)-TAG CAC CCG AGT GCT (Rox)-TTC TCC GTC TGC TCT TTT ATC TTC TC-3.

system hardware componentsThe standard system hardware components (Fig. 4, dis-cussed further below) include a programmable, expand-able-spacing pipettor, a priming and warming heater, andthe BDProbeTecET fluorescent reader. For the detection ofCT and GC, an additional lysing heater and lysing rack isincluded for specimen processing.

The expandable spacing pipettor is capable of transfer-ring samples from eight specimen tubes into microwellsin a standard 8 12 array. The pipettor is programmableand is capable of dispensing and mixing volumes up to1200 L. Aerosol-resistant tips (Matrix Technologies)were used for all transfers.

The lysing rack and lysing heater are used to heat lysespecimens for CT or CT/GC testing. The lysing rack holds96 specimen tubes and can be placed directly into thelysing heater. After the lysing step, the rack is removed toallow the samples to cool. One ergonomic feature of therack is that it permits caps on the tubes to be removedwith one hand.

The priming and warming heater contains two fixed-temperature stations capable of maintaining two distincttemperatures. Each station holds one metal microwelltray. One station, maintained at a setpoint of 72.5 C, isused in the priming step. The second station, maintainedat a setpoint of 54 C, is used to prewarm the amplifica-

tion mixture before the tray is placed into the instrument.Guide pins on this block aid in the orientation ofan adhesive sealer card, which is placed onto the pre-warmed microwell tray before transfer of the tray into theBDProbeTecET instrument.

The BDProbeTecET instrument is a fluorescent readercapable of maintaining constant temperature (52.5 C),monitoring real-time fluorescence, and reporting resultsthrough an algorithm. It consists of a heated stage capableof holding one 96-microwell tray at a time. Microwell

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trays are scanned once per minute, using fluorescentexcitation and detection through the bottom of the well.Trays are moved in one dimension on fixed rails over theoptical station. The optical station consists of an opticalbundle with eight branches, which permits an electroni-cally multiplexed interrogation of eight microwells, thuseliminating the need for two-dimensional motion of the

tray. Emitted light passes through a custom optical band-pass filter, is detected by a photomultiplier tube, and isanalyzed by software.

urine sample collection, transport, andprocessing (fig. 5)Urine specimens are collected in sterile, plastic, preserva-tive-free specimen collection cups. A urine processingpouch (Becton Dickinson Microbiology Systems) is addedto the sample cup, and the sample cup is capped. Theurine processing pouch contains a proprietary materialcapable of removing amplification inhibitors and stabiliz-ing urine specimens containing CT stored up to 6 days or

GC up to 4 days at 1830 C, or 6 days at 28 C forspecimens containing CT or GC (manuscript in prepara-

tion). At the testing site, 4 mL of urine is removed andtransferred into a 4-mL tube. After centrifugation at 2000gfor 30 min, the supernatant is decanted. Sample diluent (2mL) is added, the capped sample is vortex-mixed andplaced into the lysing rack. The rack is placed into thelysing heater (114 C) for 30 min. Samples are then re-moved from the heater and cooled for 15 min at room

temperature before use.

swab sample collection, transport, andprocessing (fig. 5)Male urethral specimens are collected using rayon swabs(MiniTip CULTURETTETM DIRECT; Becton Dickinson).For the collection of female endocervical specimens, acleaning swab is used first to remove mucus. The endo-cervical specimen is then collected with a polyurethane-tipped swab (CULTURETTE DIRECT; Becton Dickinson).Both male and female swab specimen types can betransported without preservative or liquid additive to thetesting laboratory at 230 C for up to 6 days. At the

testing laboratory, the swabs are expressed into tubessupplied prefilled with 2 mL of sample diluent. The swabs

Fig. 1. Mechanism of SDA.

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are discarded, and the expressed sample fluid is heat-lysed and cooled as described above for urine specimens.

assay procedureFor each tray of specimens assayed, one positive controland one negative control are included in the microwelltray set up and are tested like samples. Their positions are

determined by the user and appear on the plate layoutreport generated by the instrument during the login ofspecimens. A separate microwell for each control andspecimen is used for an amplification control (AC). TheAC well contains an amplifiable DNA sequence andactsto flag inhibitory specimens. Thus, for a CT test, a96-well plate will contain by one positive control (whichalso acts as a control for CT and GC primers and re-agents), one negative control, and up to 46 samples. Eachof the 48 CT wells has a corresponding AC well. Forspecimens being tested for both CT and GC, one platecontains one positive control, one negative run control,and up to 30 samples. Each of the 30 samples and two

controls requires three microwellsone for CT, one forGC, and one for AC. Once the layout of the microwells isdetermined, the test is begun.

Priming microwells are placed into their respectivemicrowell trays. Samples are lysed and then cooled atroom temperature. For a CT/GC test, 600 L of lysed andcooled specimen is aspirated from eight specimen tubessimultaneously. The pipettor tip spacing plunger is ad- justed to collapse the spacing of the tips, and 150 L isdispensed into each of three CT, GC, and AC primingwells. The remaining liquid and tips are discarded. Thisstep continues until all specimens are dispensed into thepriming tray.

The priming tray is covered and incubated at roomtemperature for at least 20 min, but for convenience inperforming multiple runs throughout a shift, trays mayincubate at room temperature for up to 6 h.

After incubation, the cover is removed from the prim-ing microwell tray. The amplification microwells areplaced in the amplification tray. The priming and ampli-fication trays are placed into their stations on the primingand warming heater for 10 min. At the end of 10 min, 100L is transferred from each microwell column in thepriming tray into the corresponding amplification micro-well column in the warming station. The pipettor isprogrammed to dispense 100 L and mix 50 L of volume

in the amplification wells three times to hydrate and mixthe sample and reagents. Tips are discarded, and the samesteps repeated until all samples and controls have beentransferred.

After the samples are transferred, a rigid self-adheringamplification sealer is applied to permanently seal themicrowells in the warming station. Paper backing isremoved from the adhesive side of the sealer and thesealer is applied smoothly to the top of the microwells.Guide pins on the warming station of the priming and

warming heater facilitate the alignment of the sealer ontothe microwell tray.

The sealed amplification tray is placed into theBDProbeTecET instrument. Amplification, fluorescencedetection, and data analysis occur in the instrument.Results are printed after the 1-h amplification and detec-tion are complete. After the completion of the assay, thesealed microwells are lifted from the trays and discardedinto a sealable plastic pouch, which provides a secondlayer of containment. The metal tray is rinsed with waterand dried before reuse.

real-time detection kinetic plotsVarious concentrations of CT (serovar LGVII, ATCC VR

902B) EBs or GC cells (ATCC 19424) were added intosample diluent and tested according to the assay proce-dure described above. Strains were obtained from Amer-ican Type Culture Collection.

clinical studiesSwab and/or urine specimens were collected from con-senting symptomatic and asymptomatic patients at acommunity family practice clinic. For endocervical andpenile urethral specimens, two swabs were collected, one

Fig. 2. Detector probe conversion during SDA.



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for testing with the Abbott LCx and one for testing withthe BDProbeTecET.

Urine specimens were divided and assayed on both theAbbott LCx and the BDProbeTecET. The results shownare the initial test results without discrepant resolution.

Results

To best understand the basis of the real-time detectionsystem, it is important to highlight the amplification anddetection mechanisms. Fig. 1 shows the two discretephases, Target Generation and Exponential Target Ampli-fication, in the mechanism of SDA. For the purpose ofsimplicity, the process is shown for only one strand of adouble-stranded target, with the understanding that thisprocess occurs on both strands and yields exponentialamplification. For the Target Generation Phase, a double-stranded DNA target (1 ) is denatured and allowed tohybridize to two primers, B1 and S1 (2 ). The B1 is a bumper primer, whereas the S1 primer contains thesingle-stranded restriction enzyme sequence 5 to a target-

binding region of the primer. In the presence of the BstDNA polymerase and dNTP mixture, simultaneous ex-tension products of B1 (3 ) and S1 are generated (4 ). Thisprocess displaces the S1 products, which may then behybridized to the opposite strand primers, B2 and S2 (5 ).The simultaneous extension of both primers producesspecies 6. Species 6 is capable of carrying out the Expo-nential Target Amplification Phase. Species 6 is a sub-strate for the BsoBI enzyme in the mixture, which recog-

nizes the appended double-stranded BsoBI site. This BsoBIsite contains a thioated dCTP nucleotide incorporatedduring the Target Amplification Phase, which makes thesite refractory to double-stranded cleavage by the en-zyme. Instead, the strand lacking this modified dC withinthe restriction site is nicked by the BsoBI (7 ). The Bst DNApolymerase next binds to this nick and begins the synthe-

sis of a new strand while simultaneously displacing thedownstream strand (810). This step recreates the double-stranded species 7, and the iterative nicking and displace-ment process repeats. The displaced strands are capableof binding to opposite strand primers, which producesexponential amplification at 52.5 C. These single-stranded products also bind detector probe for real-timedetection.

Present in the SDA reaction is a single-stranded DNAprobe containing fluorescein and rhodamine labels (seeMaterials and Methods). The region between these labelsincludes a stem-loop structure. The loop comprises arecognition sequence for the BsoBI enzyme. This probe

also contains a target-specific sequence 3

to the rhoda-mine label. Before specific target amplification by SDA,the fluorescein and rhodamine labels are proximal to eachother such that any excitation of the fluorescein leads totransfer of the emitted energy to the rhodamine label. Thenet effect is that very little emission from excited fluores-cein is detected. After SDA, the probe is converted to adouble-stranded species, which is cleaved by the BsoBIrestriction enzyme. This cleavage causes the physical

Fig. 3. Mechanism of fluorescence ET produced from a single-stranded probe during SDA ( A) and real-time detection of lysed CT EBs and GC cells (B).



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separation of the fluorescein and rhodamine labels suchthat no ET from the excited fluorescein to rhodamine can

occur. The net effect is that emission is detected from anexcited fluorescein label, and this emission is indicative ofspecific amplification attributable to the presence of the

target sequence.The specific steps for the conversion of this fluorescent

single-stranded probe are shown in Fig. 2. In step 1, adisplaced strand (thick line), the probe containing fluo-

rescein (E) and rhodamine (F), and an amplificationprimer hybridize. The simultaneous extension by theDNA polymerase of both the amplification primer and

probe leads to displacement of an extended probe (2 ). Instep 3, the extended probe binds the opposite strand

primer and is extended (4 ). This extension creates adouble-stranded BsoBI site, which is flanked by both the

fluorescein and rhodamine labels. This extension stepcreates a BsoBI site that lacks thioated dCTP incorporationat the nucleotide position of BsoBI cleavage. As a conse-

quence, binding of BsoBI at the site causes double-stranded cleavage instead of nicking (5 ); the two labels

thus are physically separated, and emission of fluoresceinis detected. These steps occur simultaneously during the

SDA process. This detection process is distinguished fromTaqman in several ways, including the fact that Taqman

uses PCR, thus exploiting the 5-3 exonuclease activity of

the Taq polymerase, whereas SDA uses exonuclease-freepolymerase, and that the Taqman method requires ther-

mocycling for the formation of fluorescent product.An overview of the fluorescence ET detection process

that occurs during SDA is presented in Fig. 3A. As seen in

the kinetic plots of Fig. 3B, the formation of these fluores-cent products is both continuous and rapid. For both CT

and GC, the detection of small numbers of cells or EBsoccurs within 1 h. The doseresponse seen in Fig. 3B

allows for the potential application of target quantifica-tion on this system.

The components of the system instrumentation and theworkflow are shown in Figs. 4 and 5. The workflow isdesigned to allow for multiple analytical runs within a

single shift and optimal time to first results. For example,96 specimens and controls can be lysed at one time. This

provides sufficient specimens for two cycles of CT assays,or three cycles of CT/GC assays. Lysed specimens may

cool from 15 min to 6 h. Once specimens are dispensedinto the priming microwells, they may be incubated up to6 h. Thus, specimens sufficient for additional analyses

may be lysed, primed, and staged for the priming andwarming, and detection steps. With six analytical runs

achievable in a single shift, up to 180 CT/GC combinationtests or up to 276 CT assays can be performed. This AC

feature can be deselected by the user, if desired, thus

Fig. 4. Hardware components of the BDProbeTecET system.



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allowing 94 wells for CT detection per analytical run. Forother analytes under development, the instrument iscapable of monitoring two amplification reactions in onewell, allowing for multiplex test results, such as ananalyte and internal control. In both of these cases, athroughput of up to 564 patient results can be achievedper shift.

The detection of CT and GC from clinical samples isshown in Tables 1 and 2. The algorithm resident in theinstrument reports results as positive, negative, indeter-minate (inhibited AC), or equivocal on the basis of prese-lected cutoff values established using clinical specimens.The reportable results shown in Tables 1 and 2 do notinclude indeterminate (two urine specimens each in theCT assay) or equivocal specimens (two swabs in the CTassay and one in the GC assay), which must be retested.The Abbott LCx does not have an AC, and thus cannotreport indeterminate results. Because indeterminate andequivocal results are not reportable results, they are notincluded into calculations on sensitivity or specificity. Thesensitivity and specificity for reportable results using the

BDProbeTecET CT and GC assays relative to the AbbottLCx are 100%.

Discusssion

Although a limited number of specimens were tested, thedata show that excellent performance can be achievedwith the system when patient swab and urine specimensare used. Complete characterization of system perfor-mance, including clinical trial data, will be publishedseparately (manuscript in preparation). Separate internal

studies (data not shown) of interfering substances showthat the presence of up to 2% blood (swabs) does notaffect results. One of the reasons for this is that the effectof such potentially interfering substances is mitigated bythe real-time data acquisition and metrics applied by thealgorithm. For example, the real-time detection permitssubtraction of signals resulting from fluorescent sub-stances. This is a distinct advantage in a clinical setting,where such substances may be encountered in specimens.

Contamination with amplicons or target DNA is alsominimized with the system design. The controlled dis-pense and aspirate programs on the pipettor preventsplattering in the transfer steps. Aerosol-resistant tipsprevent sample-to-sample carryover. Because no amplifi-

cation occurs in the priming wells, no amplicon contam-ination is possible at this stage. The sealed microwells and

Fig. 5. Workflow overview for the preparation of specimens, sample lysis, priming and warming, and amplification and detection.

Table 1. Detection of CT in clinical samples.

BDProbeTecET

LCx

Positive Negative

Positive 10 0

Negative 0 47

Total 10 47

Table 2. Detection of GC in clinical samples.

BDProbeTecET

LCx

Positive Negative

Positive 1 0

Negative 0 57

Total 1 57



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subsequent disposal into sealed plastic pouches providestwo levels of containment from amplicon release. Thusoverall, the system approach has been to design outcross-over and contamination potential. Internal studieson cross-contamination, where high-positive and low-positive tubes are interspersed, demonstrate 0.1% (1 of960) cross-contamination and validate that this design out

approach has met with success.

In conclusion, the BDProbeTecET system, which is basedon real-time fluorescent detection and SDA, provides anew technology for the clinical setting. The system hassuperior throughput and time to first results, simpleworkflow, and minimal risk for release of ampliconcontamination.

We thank Judith Lovchik of the University of Maryland,Baltimore, MD, for testing the BDProbeTecET and AbbottLCx systems on clinical specimens. We also thank Neil

Jensen for providing the kinetic plots and Francois Guillotfor workflow graphics. The fluorescent probe design andreal-time probe conversion reactions depicted in Fig. 2were conceived by J.G. Nadeau, J.B. Pitner, J.L. Schram,and C.P. Linn [European patent application (1998). Pub-lication no. 0 881 302].

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Michael C. Little et al- Strand Displacement Amplification and Homogeneous Real-Time Detection Incorporated in a Second-Generation DNA Probe System, BDProbeTecET