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1
Advanced Diagnostics Group, Dade Behring Inc., PO Box 49013, San Jose, CA 95161-9013.
a Author for correspondence. Fax 408-239-2707; e-mail samuel_rose{at}dadebehring.com
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Abstract |
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Methods: Luminescent oxygen channeling assay (LOCITM) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types.
Results: In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets.
Conclusions: This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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200 nm (approximately the diameter of a LOCI particle itself)
for chemiluminescent signal to be generated. The close proximity of
particle pairs causes photochemically triggered chemiluminescence to be
emitted in an otherwise very dark background, producing
high-sensitivity assays. The particles are usually present in an assay
at much lower concentrations than used in latex agglutination; hence,
adventitious close proximity of the sensitizer and chemiluminescent
particles in a LOCI assay is rare.
One of the two particle populations has a photosensitizer dissolved in
it that produces singlet oxygen when exposed to light. The second
population of particles has dissolved within it an olefin that will
produce a chemiluminescent emission upon reaction with singlet oxygen.
Because the lifetime of the singlet oxygen reactive species in water is
very short (4 µs), the sensitizer and chemiluminescent LOCI
particles need to be essentially bound to one another to produce a
signal. Both particle types have a hydrogel coating that reduces
nonspecific interactions with assay matrix components and also provides
for a surface that can be functionalized with appropriate binding
moieties. Detection of the LOCI signal is accomplished by alternately
exposing the PCR tube to a laser light source and then to a
photomultiplier tube to sum the chemiluminescent decay (2).
Adoption of the homogeneous LOCI technology for non-nucleic acid applications is well under way. The appeal of LOCI qualities such as simplicity, speed, high sensitivity, and broad applicability is compelling. Application of LOCI technology for both the clinical diagnostics and research market is a focus of much effort, and it has recently been commercialized for high-throughput drug screening (AlphaScreenTM; Packard BioScience).
As the name implies, the first application of LOCI was for immunoassays. We have discovered that the LOCI principle can be applied to the detection of nucleic acids as well. These applications include quantified homogeneous PCR combined with LOCI in an endpoint determination and also in a "real-time" fashion. We report here on the progress of these studies combining PCR amplification with LOCI detection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) was prepared by modifications of a published method
(4). The first step of the synthesis was modified to
incorporate tetra-t-butyl groups into the final product.
This was designed to reduce stacking and, hence, self-quenching as was
observed in the case of phthalocyanine. The
6-t-butyl-1,3-diiminobenz(f)isoindolin was
prepared according to the published method (4) for
the preparation of 1,3-diiminobenz(f)isoindolin, except that
the 6-t-butyl-2,3-dicyanonaphthalene precursor was used in
place of 2,3-dicyanonaphthalene. The remainder of the synthesis
followed the published procedure using this modified precursor.
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The latex particles used for dyeing were carboxylate-modified polystyrene, 203 ± 4 nm in diameter with 0.09 milliequivalents of carboxyl per gram of beads. A 10% suspension in water was obtained from Seradyn (Indianapolis, IN). The chemiluminescent particles ("dopTAR" particles) used in these studies were dyed with thioxene according to a previously described procedure (1). The organic dyes 1-chloro-9,10-bis(phenylethynyl)anthracene and rubrene were used instead of europium and phenanthroline. These dyes shift the thioxene chemiluminescent emission to a longer wavelength. Heptadecylbenzene was incorporated into the chemiluminescent particles as a plasticizer to improve the linearity of decay kinetics. This was readily accomplished subsequent to the dyeing procedure (1) by the addition of a 10 mL/L solution of the heptadecylbenzene in ethanol (95 °C) to a 20 g/L suspension of the TAR beads in 100 mL/L ethanol in water (95 °C). After 20 min, the beads were cooled, the ethanol concentration was increased to 750 mL/L, and the beads were centrifuged. The resuspended pellet could then be enhanced by surface modification (1).
Oligodeoxynucleotides from Oligos Etc. were coupled to
the latex following a modification of the procedure of Ullman et al.
(2). The cross-linking agent succinimidyl
6-((iodoacetyl)amino)hexanoate was substituted for the
sulfo-SMCC used by Ullman et al. (2), and the
oligomers were thiolated at the 3' end. The thiolated oligomer
contained a 3'
sulfo-[succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate],
OPO2O(CH2)3SS(CH2)3OH,
introduced from the corresponding phosphoramidite during synthesis.
Free thiol was generated before coupling as described previously
(2). Binding capacity was determined by incubating the beads
with a small excess of fluoresceinated complementary oligo (Oligos
Etc.) for 30 min at 37 °C, centrifuging to remove the beads, and
determining fluorescence in the supernatant. Typically, 5000
oligonucleotides were bound to each particle.
Purified and quantified Chlamydia trachomatis elementary bodies were from the Syva Division of Dade Behring Inc., and total DNA was extracted by standard phenol-chloroform protocols. Mycobacterium tuberculosis (M. tb.) genomic DNA (GenBank Accession No. Y14045NID) was the kind gift of Dr. Chris Green, SRI International, Menlo Park, CA.
Assay sensitivity was assessed by calibration curves using as analytes synthetic oligonucleotides of known concentration to link the two probes in solution, followed by binding of the complex to the beads. The signal-to-background ratio for 1 pmol/L analyte was 6.0 for phthalocyanine beads irradiated at 680 nm and 6.2 for naphthalocyanine beads irradiated at 780 nm.
Endpoint PCR/LOCI.
The PCR amplification protocol for LOCI
endpoint analysis was as follows. The repetitive insertion sequence
IS6110 in the genome of M. tb. was used as the target for
PCR. The forward primer was LB-3 (5'-CGA TCG AGC AAG CCA TCT-3'), and
the reverse primer was ZL-4 (5'-ACT GGT AGA GGC GGC GAT GGT T-3'),
producing a 139-bp amplicon. The phthalocyanine sensitizer LOCI
particle was decorated with 5'-(AGTA)6-3'
oligomers, the naphthalocyanine sensitizer particle with
5'-(ATAG)6-3' oligomers, and the chemiluminescent
dopTAR particle with 5'-(dA)24-3' oligomers. The
oligomer probe that linked the target sequence to the phthalocyanine
sensitizer particle was ZN-1 [5'-ACG GAT AGG GGA TCT CAG TA
(TACT)5-X-3']. The linking oligomer for the
naphthalocyanine sensitizer particle to the internal control sequence
was IC [5'-GAC AGT GTA GAT AGA TGA CAG TCG
(CTAT)5-X-3']. The probe common for linking both
the target and the internal control amplicon to the chemiluminescent
dopTAR particle was ZL-5 [5'-(T)20 GCG TAC TCG
ACC TGA AAG AC-X-3']. X indicates a 3' C-7 amino elongation blocking
group at the 3' terminus of the probes.
An internal control amplicon was created for the endpoint PCR
quantification. This was made using a PCR protocol for the synthesis of
a "mutant fusion product" amplicon (5), which was the
same length as the amplicon of the target M. tb. DNA and
amplified by the same primers but which had a 24-base sequence
substituted within it. This 24-base sequence (5'-GAC AGT GTA GAT AGA
TGA CAG TCG-3') was complementary to the IC probe described above (see
Fig. 2
). There was no detectable difference in the efficiency of the
PCR amplification of the target M. tb. DNA and the internal
control amplicon.
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The reaction mixture consisted of 4 µL of target M. tb.
DNA or water, 5 U of cloned Pfu exo+ polymerase
(Stratagene), 200 µmol/L each dNTP, 250 nmol/L each PCR primer, 10
mmol/L Tris-HCl (pH 8.3), 4 mmol/L MgCl2, 50
mmol/L KCl, and 0.2 g/L bovine serum albumin. Probes ZN-1, IC,
and ZL-5 were each present at a final concentration of 50 nmol/L, and
3.75 µg of the chemiluminescent particle and 2.5 µg each of the
sensitizer phthalocyanine- and naphthalocyanine oligomer-decorated
particles were included in the reaction. Typically, 20 000
molecules of the control amplicon were added to each reaction. PCR
cycling conditions were as described below.
Real-time PCR/LOCI.
PCR amplification for real-time analysis
was as follows. Total DNA from C. trachomatis elementary
bodies containing the cryptic plasmid target was purified and used as
the target. It was assumed that there were 10 cryptic plasmid
molecules per elementary body (6). The forward primer used
was FP2 (5'-GGA CAA ATC GTA TCT CGG GTT AAT-3'), and the reverse primer
was RP2 (5'-GGA AAC CAA CTC TAC GCT GTT-3'), which produced a 518-bp
amplicon. The probe that linked the phthalocyanine sensitizer particle
to the amplicon was SP2 [5'-CGC TGC GAA TAG AAA AAG TCC A
(T)20 X-3']. The probe that linked the
chemiluminescent particle to the amplicon was PN2
[5'-(TACT)5 GCC TAG CTG CTA TAA TCA CGA X-3'].
X indicates a 3' C-7 amino elongation blocking group at the 3' terminus
of probes. Sensitizer particles were labeled with 5000
5'-(dA)24-3' DNA oligomer molecules, and the
chemiluminescent particles were also labeled with 5000
5'-(AGTA)6-3' DNA oligomer molecules.
The reaction mixture consisted of 5 µL of DNA target or water, 1 U of Pfu exo- polymerase (Stratagene), 200 µmol/L each dNTP, 250 nmol/L each primer, 10 mmol/L Tris-HCl (pH 8.3), 4 mmol/L MgCl2, 50 mmol/L KCl, and 0.2 g/L bovine serum albumin. Probes SP1 and PN2 were present at a final concentration of 50 nmol/L, and 2.5 µg each of the sensitizer and chemiluminescent oligomer-decorated particles was included in the reaction. The final reaction volume was 50 µL. Cycling conditions were as described below.
instrumentation
After PCR amplification, the LOCI signal was typically read from
reaction volumes of 40–100 µL contained in 0.2-mL polypropylene
"eight-strip" tubes (ISC Bioexpress) using a custom-built
breadboard instrument.
The elements constituting the reader used in the chemiluminescent assays were as follows: two lasers for reaction mixture illumination; an illumination chamber; optical train with collimating lenses, filter wheel, and shutter; photomultiplier tube (PMT); controlling computer; and software.
The reaction mixture is illuminated by two laser diodes. The illumination wavelengths selected were 680 nm and 780 nm. The lasers’ respective powers were 30 and 70 mW. The diodes are contained inside a holder, which acts as a heat sink and holds a focusing lens. The lens is adjusted to provide an illumination spot approximately the size of the reaction mixture at the bottom of the strip tubes (a few mm in diameter). Both laser assemblies are focused on the same spot, the bottom part of the vial containing the reaction mixture. The excitation current of each laser can be set independently. Illumination times and sequences are controlled by the computer.
The illumination chamber holds the reaction vial in the center of a white Teflon reflector. The purpose of the reflector is to improve illumination efficiency and light collection. The light axis of the laser diode is perpendicular to the light collection system axis.
The optical train uses two aspherical lenses to collimate and focus the light on the PMT photocathode. A shutter and a filter wheel are inserted between the two lenses. The shutter is used to protect the PMT from overexposure and potential damage during the illumination sequence. The filter wheel allows for automatic selection of the proper filter depending on the dye excited.
The filter wheel contains up to five different filters. The filters are selected to let the emission wavelength go through and block any potential emission from unwanted sources and allow proper selection of the emission wavelength.
A PMT is used to detect the chemiluminescence emission. The tube works in the photon-counting mode. Pulses are counted by a counter board installed in the controlling computer.
Special software has been written to control the hardware and acquire the data.
The temperature of the tubes in the reader was maintained at 37 °C. Specific LOCI detection protocols are described with each of the assays below.
Thermocycling platforms included the Ericomp TwinBlockTM System (Ericomp) and the Uno and T3 instruments (BioMetra).
assays
PCR/LOCI DNA quantification assay: endpoint analysis.
The
probes that linked the oligomer LOCI particles to the PCR-amplified DNA
were specific for each of the two sensitizer particles and for the
chemiluminescent particle (Fig. 2
). A PCR amplicon internal
quantification control was created that contained a substituted
24-base-long sequence specific for the naphthalocyanine sensitizer
particle/probe. This control amplicon was of the same length and was
amplified by the same primers as the target polynucleotide
(7).
The PCR amplification consisted of an initial denaturation temperature of 95 °C for 3 min, followed by 36 cycles of the three-temperature regimen of 95 °C for 20 s, 63 °C for 1 min, and 73 °C for 1 min. After 5 min at 75 °C to complete DNA polymerization, the double-stranded amplicon was denatured at 95 °C for 2 min, cooled to 50 °C for 15 min to allow probe annealing to the amplified DNA, and finally cooled to 37 °C for 60 min to allow annealing of the probes to the LOCI oligomer particles.
The target signal (phthalocyanine sensitizer dye particle) was obtained by illuminating the reaction tubes with a 678 nm laser for 1 s followed by reading the chemiluminescent decay signal for 1 s. This was followed by a 10-s delay to allow the entire chemiluminescent signal to decay completely. The control amplicon signal (naphthalocyanine sensitizer dye particle) was then obtained by illuminating with a 780 nm laser for 1 s and reading for 1 s, and repeating this illumination/reading protocol six times. The total photon counts from each illumination type were summed. After subtraction of the counts from the no-target control, the ratio of the total counts between the two illuminations allowed quantification of the target DNA.
PCR/LOCI DNA quantification assay: real-time analysis.
In
principle, annealed complexes consisting of DNA LOCI particle pairs,
probes, and single-stranded target DNA such as shown in Fig. 2A
can be
allowed to form at any time during PCR amplification. All that is
required is sufficient cooling of the reaction to a temperature low
enough for stable base pairing to allow creation of the two-bead LOCI
complex and sufficient time to form enough complexes to give a
detectable LOCI signal.
A PCR/LOCI homogeneous amplification of a Chlamydia cryptic plasmid target was amplified for 10 cycles by a standard three-temperature protocol of 95 °C for 15 s, 62 °C for 1 min, and 74 °C for 1 min. On the 10th cycle, the reaction was cooled to 37 °C for a total of 2 min immediately after the 95 °C denaturing temperature. At the end of 2 min, the samples were irradiated using a 675-nm laser for 0.1 s followed by 1 s of photon counting, and this was repeated three times. Total photon counts were summed. After this LOCI reading, cycling was resumed by heating to 62 °C and holding for 1 min, then heating to and holding at 74 °C for 1 min, followed by a 15-s 95 °C denaturation step. On every second cycle (e.g., cycle 12, 14, and 16), the 37 °C cooling step was introduced to allow another LOCI reading.
Most of these experiments were performed manually, moving the strip tubes from a thermocycling block to a LOCI detector at the appropriate PCR cycle. Some of the assays utilized an instrument custom designed and built at Dade Behring, which automated this process. In this case, a mechanical XZ arm would lift the strip tubes from the thermocycling block and put them in a LOCI reader, and then retrieve the tubes and place them back in the thermocycling block. Data were stored in a Microsoft Excel spreadsheet.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). LOCI sensitizer particles were of two types. One of
the particles incorporated phthalocyanine and the second
naphthalocyanine as the singlet oxygen generators. These were
separately directed to the target and quantification control by
specific probes. Different illumination wavelengths separately
stimulated singlet oxygen production from these two sensitizer
particles. The chemiluminescent particle population was able to anneal
to both control and target amplicons.
Previous studies have shown that PCR amplification, even after reaching
a plateau, can be quantified if internal competitive controls amplify
with the same efficiency as the target (8). In these
experiments a fixed and known concentration of a control amplicon was
introduced into each PCR reaction before amplification. The target DNA
(Chlamydia cryptic plasmid) was introduced into the same PCR
reactions over a range of known concentrations, and the amplification
proceeded for 40 cycles. At the conclusion of amplification, the tubes
were placed in a breadboard LOCI reader. Two different wavelength
illuminations allowed the specific sensitizer/chemiluminescent LOCI
bead pairs to distinguish between control and target amplicons (Fig. 2
). When the input control amplicon concentration was known,
the ratio of the signals from these separate illuminations and LOCI
signal readings allowed determination of the unknown target amplicon
over a three-log dynamic range (Fig. 3
.)
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pcr real-time quantification
If an ongoing PCR reaction containing LOCI particles and probes is
cooled to allow formation of an annealed complex as in Fig. 2A
, then a
LOCI signal can be obtained at any desired point in the DNA
amplification. We added a 37 °C cooling step with a LOCI reading to
the PCR amplification starting at the 10th cycle and every second cycle
thereafter (Fig. 4
). The signals obtained were compared with the LOCI signal from
a zero target control reaction. The first PCR cycle that produced a
signal 10 SD above the no-target control background was recorded. The
cycle at which this occurs is called the "threshold cycle". A plot
of this cycle number against the log of the number of DNA targets
produced a straight line that covered six orders of magnitude of target
concentration (Fig. 5
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The targets of C. trachomatis and M. tb. DNA were chosen merely as models for quantification assays. We had previously optimized qualitative PCR assays using these targets and decided to use them for testing quantification feasibility. Usually, there is no clinical need to quantify these organisms. Preliminary reverse transcription-PCR/LOCI endpoint quantification studies of HIV 1 in our laboratory are showing good progress.
Homogeneous real-time detection and quantification of DNA targets by PCR was first achieved using the DNA intercalator dye ethidium bromide, where increasing fluorescence detected the amplifying double-stranded DNA (9). The more recent TaqMan technology (10) added specificity to real-time PCR quantification because only the desired amplification product is detected. As in TaqMan, we have also used the concept of a threshold cycle to quantify the target DNA in our real-time assays. A six-log dynamic range could be achieved.
The cooling of the PCR reaction to 37 °C in the real-time detection
scheme could be problematic if mispriming was to occur at such a low
temperature. We have not explored the possibility of merely lengthening
the oligomers conjugated to the LOCI particles and the probes to raise
the LOCI reading temperature. However, we have found that when using a
hot-start procedure for priming specificity, the full range of
detection shown in Fig. 5
can be achieved even in the presence of 1
µg of human DNA (data not shown).
Sensitizer and chemiluminescent LOCI oligomer particles have been shown
to survive the typical thermocycling profiles of PCR. After 40
thermocycles of a three-temperature protocol of 15 s at 95 °C,
1 min at 68 °C, and 1 min at 72 °C, sensitizer and
chemiluminescent DNA LOCI particles each lost only 20% of their
maximum potential signal output compared with nonthermocycled DNA LOCI
particles (data not shown). This result was consistent across several
preparations of the DNA-conjugated LOCI particles. The degradation in
LOCI signal generation capability attributable to thermocycling did not
have a material effect on the assay because the numbers of amplicons
produced by target amplification were orders of magnitude above the
detection limit of the DNA LOCI particles.
The shapes of the curves in Fig. 4
are unusual. The maximum signals
achieved are highest with the higher concentrations of starting target
DNA and decrease as the target DNA concentrations decrease. The signals
grow to a maximum, and then gradually decrease with repeated cycles and
LOCI readings. In investigating this phenomenon, we found that repeated
exposure to singlet oxygen, through the repetitive illuminations at
every second thermocycle of the PCR, gradually decreased the ability of
the hybridized LOCI complex to generate chemiluminescence. Current
experiments suggest that the probes that anneal to the sensitizer
particles are being inactivated, most likely because of singlet oxygen
destruction of guanosine residues. Preliminary experiments in which
there is substitution of inosine for guanosine in the sensitizer probes
indicated that the use of inosine substantially reduces this loss of
chemiluminescent signal (data not shown). It should be noted
that even without this improvement it was possible to detect small
numbers of DNA targets.
An advantage of using LOCI particles for detecting amplifying DNA is the relatively simple requirement of merely synthesizing probes that anneal to the specific amplicon and to the oligomers on the particles. Designing a probe that will be specific for the amplicon(s) is trivial. This represents a much easier design requirement than the sometimes problematic need of, for example, designing a TaqMan probe that works well with the combination of specific PCR amplification conditions, an appropriate sequence within the amplicon, and the 5' nuclease activity of Taq DNA polymerase.
For real-time detection using LOCI, the necessity to lower the temperature of the PCR reaction to 37 °C to form a detectable particle complex substantially slows the overall assay. Making the probes longer so that the temperature of complex formation could be raised, decreasing the examination of the LOCI signal to every third or fourth cycle instead of every second cycle, or increasing the kinetics of LOCI bead pair formation (e.g., more beads or more probes) needs to be explored. The real-time PCR LOCI assay in its current state of development is probably not suitable for the very fast thermocycling machines now commercially available. The endpoint assay should perform very well, but we have not yet built a chemiluminescent fast-cycler to test this opportunity.
In conclusion, we have demonstrated that LOCI particles conjugated with oligodeoxynucleotides are capable of quantifying DNA targets in either endpoint or real-time formats. For the endpoint assays, two signals could be detected in a homogeneous format, which can allow an unknown target concentration to be determined by its ratio to a known internal control signal. In a real-time detection protocol, repeated LOCI readings can be used to determine a threshold signal that is directly related to the DNA target concentration. Both protocols are homogeneous closed-tube methods with the dual advantages of controlling PCR carryover contamination and easier automation.
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Acknowledgments |
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Footnotes |
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2 Present address: Targesome, Inc., 4030 Fabian Way, Palo
Alto, CA 94303.
3 Nonstandard abbreviations: LOCI, luminescent oxygen channeling assay; M. tb., Mycobacterium tuberculosis; and PMT, photomultiplier tube.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  J. Hynes, S. Floyd, A. E. Soini, R. O'Connor, and D. B. Papkovsky Fluorescence-Based Cell Viability Screening Assays Using Water-Soluble Oxygen Probes J Biomol Screen, June 1, 2003; 8(3): 264 - 272. [Abstract] [PDF]
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 L. Beaudet, J. Bédard, B. Breton, R. J. Mercuri, and M. L. Budarf Homogeneous Assays for Single-Nucleotide Polymorphism Typing Using AlphaScreen Genome Res., April 1, 2001; 11(4): 600 - 608. [Abstract] [Full Text]
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 A. Dafforn, H. Kirakossian, and K. Lao Miniaturization of the Luminescent Oxygen Channeling Immunoassay (LOCITM) for Use in Multiplex Array Formats and Other Biochips Clin. Chem., September 1, 2000; 46(9): 1495 - 1497. [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
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