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1
Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
2
OraSure Technologies Inc., Bethlehem, PA 18015.
aAuthor for correspondence. Fax 31-71-5276180; e-mail Corstjens{at}lumc.nl.
Abstract
Background: A lateral-flow (LF) device using the new reporter up-converting phosphor technology (UPTTM) was applied to DNA (hybridization) assays for the detection of specific nucleic acid sequences, thereby aiming to perform the test outside well-equipped laboratories. The methodology reported here is sensitive and provides a rapid alternative for more elaborate gel electrophoresis and Southern blotting. In a preliminary study, it was applied to screen for the presence of human papillomavirus type 16 (HPV16) in a defined series of cervical carcinomas.
Methods: A LF assay was used to capture haptenized DNA molecules and hybrids, which were immunolabeled (before LF) with 400-nm UPT particles. These particles emit visible light after excitation with infrared in a process called up-conversion. Because up-conversion occurs in only the phosphor lattice, autofluorescence of other assay components is virtually nonexistent.
Results: The use of the UPT reporter in LF-DNA tests, as compared with colloidal gold, improved the detection limit at least 100-fold. UPT LF-DNA tests were successfully applied to detect (in a blind test) the presence of HPV16 in DNA extracts obtained from cervical carcinomas. Test results matched 100% with previous characterization of these carcinomas.
Conclusions: The use of UPT in LF assays to detect specific nucleic acids provides low attamole-range sensitivity. Hybridization and consecutive detection of PCR-amplified HPV16 sequences were successful in a background of 10 µg of fish-sperm DNA. The sensitivity of UPT detection in these complex mixtures indicates that detection of viral infections without PCR or other amplification technique is achievable.
Improving the performance of diagnostic assays is an ongoing challenge (1)(2). Besides standard performance indicators such as sensitivity, specificity, and reproducibility, factors such as speed and related costs have become increasingly important. In this context, lateral-flow (LF) 1 assays have an acknowledged position and are well suited for rapid onsite testing. Most LF tests reported to date relate to immunodiagnostics and are based on the specific interaction between antigens and antibodies (3). In situations where diagnostic information is best supplied at the nucleic acid level, however, there is a need for rapid hybridization-based assays (4). For example, in case of HPV diagnostics, antibody-based methods provide insufficient specificity or sensitivity, and more detailed genotyping is therefore indicated (5).
Despite the demand for rapid hybridization-based tests, only a few LF-type assays have been described to detect specific nucleic acid sequences (6)(7). In these systems, the actual hybridization reaction is generally performed before the flow. Subsequent capture of the hybrid (in LF) is based on the formation of hapten-antibody [e.g., digoxigenin-anti-digoxigenin (DIG-
DIG)] or hapten-protein (e.g., biotin-avidin) complexes. An important reason to apply hapten capture instead of hybridization-capture is that the kinetics of hybridization in LF are quite different and much more complex as compared with the formation of the antigen-antibody complex used in common immunochromatography assays.
The few described LF-DNA systems are convenient with respect to simplicity, speed, reproducibility, and costs. Thus they offer a good alternative to more elaborate gel electrophoresis. However, an obstacle in the further development of LF-DNA systems is the lack of sufficient sensitivity to successfully compete with assays that use nucleic acid-based amplification techniques, such as PCR or comparable assays (8). Although single-molecule detection without the use of target amplification has been reported (1), current methods are not yet suited for routine clinical applications. Depending on the reporter molecule used, the currently available LF-DNA systems have a detection limit of 
1 fmol. This is one order of magnitude better than the commonly used staining of gels with ethidium bromide, but not sufficient to pursue the development of diagnostic tests without a target amplification step.
The application of UPTTM as reporter in immunochromatographic assays (9)(10) and nucleic acid microarrays (11) provides 10–100-fold better detection limits than fluorescent reporters. Therefore, we anticipated that the application of UPT in LF-DNA systems will lead to better detection sensitivities, thereby achieving simple, rapid, and sequence-specific DNA tests independent of target amplification.
Up-converting phosphors are particles (400 nm) that are composed of rare earth lanthanide elements embedded in a crystal. These particles exhibit anti-Stokes behavior by up-converting low-energy, infrared (IR) light (980 nm) to high-energy, visible light (12). The emission spectrum obtained is mainly dependent on the embedded rare earth elements (13); phosphors with distinct green, red, and blue emission spectra are available.
The phosphor particles used in this study emit green light (550 nm) and are composed of ytterbium and erbium as respective absorber and emitter ions embedded in a preceramic matrix. The UPT phosphor particles have an unmatched contrast because up-conversion of IR light to visible light is restricted to the crystal lattice. Therefore, autofluorescence after IR excitation of other assay components, including samples, is entirely absent. Because detection of a single phosphor particle has been demonstrated, the sensitivity is mainly determined by the degree of nonspecific binding. As a model, but also because of its large diagnostic potential with the use of LF assays, we tested UPT for the detection of human papillomavirus type 16 (HPV16) DNA. HPV16 and other high-risk HPV types are considered sensitive screening markers for the detection of cervical cancers.
The association of HPV with malignancy of the uterine cervix is well established (5)(14). More than 80 types of HPV have been identified and are commonly divided in two classes: the high- and low-risk types. HPV16 (as well as HPV18, -31, -33, and -45) is one of the most common high-risk types (15). The target DNA used in this study consisted of a 452-bp DNA fragment PCR-amplified from gene L1 (16) of HPV16.
Two variants of a UPT LF-DNA test were developed: (a) a very sensitive test intended to detect HPV16 DNA for rapid prescreening and (b) a secondary hybridization-based confirmatory test. Standard PCR is applied to amplify HPV16 DNA if present; this is subsequently detected with UPT LF. Positive prescreening results may then be verified in a second hybridization-based, confirmatory test. Both tests were developed with the use of genomic DNA from an HPV16 cell line (CaSki; ATCC no. CRL-1550). The specificity and sensitivity of the two tests are discussed. The UPT LF-DNA test system was applied in blind tests on DNA samples obtained from 10 cervical carcinomas, of which 6 were known to be HPV16 positive, 3 others were characterized previously as HPV18, and 1 as HPV45.
Materials and Methods
cervical carcinoma samples and (test-evaluation) control cell lines
A cell line containing integrated HPV16 and a HPV-negative cell line were obtained from ATCC. DNA extracted from the HPV16-positive CaSki cell line (ATCC no. CRL-1550; human cervical epidermoid carcinoma) and the HPV-negative 143B cell line (ATCC no. CRL-8303; human osteosarcoma; TK-) was used for the development and evaluation of the test systems.
DNA was extracted from 10 confirmed cases of uterine cervix carcinoma selected from the archive of the Department of Pathology, Leiden University Medical Center. Of these cases, six were previously characterized as matching HPV16, three as HPV18, and one as HPV45.
construction of the lf strips
LF strips were manufactured by mounting the following on a plastic backing (Fig. 1
): a 20-mm glass-fiber sample pad (glass-fiber no. 33; Schleicher & Schuell, Inc.); a 45-mm, laminated Hi-Flow nitrocellulose membrane (SRHF04000; Millipore); and a 20-mm paper absorbent pad (paper no. 470; Schleicher & Schuell). Using a Linomat IV striper (CAMAG Scientific Inc.), we provided the nitrocellulose membrane with the following: a target-capture line of Avidin-D (Vector Laboratories Inc.); a flow-control line of goat anti-mouse IgG antibody (Rockland Immunochemicals); and an index line of plain phosphor particles as the standard for luminescence. Avidin-D diluted in a Tris-HCl buffer (10 mmol/L, pH 8.0; 10 mL/L methanol) was loaded at a density of 720 ng per 4 mm. Goat anti-mouse IgG antibody diluted in the same Tris-HCl buffer was loaded at a density of 500 ng per 4 mm, and the index line was prepared at a density of 250 pg per 4 mm. Filters were allowed to dry at room temperature (RT) for 30 min after application of the target, control, and index lines. An automatic cutter (Index Cutter-I; A-Point Technologies) was used to cut strips 4 mm in width. The strips were stored dry at RT. Different batches (40 strips each) were kept apart.
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Commercially available LF strips with gold detection (DNA Detection Test Strips) were used according to the manufacturer’s description (Roche Diagnostics Netherlands).
production of the upt reporter conjugate
All UPT detections were performed indirectly with phosphor-conjugated monoclonal antibody directed against the DIG hapten [(MDIGPHOS); Roche Diagnostics Netherlands). We coupled the MDIG antibodies to silicated, C10-carboxylated phosphor particles [diameter, 400 nm; for further specific details of the physical and chemical properties of the up-converting phosphor particles see Ref. (12)] using a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride-mediated reaction. Phosphor particles were first activated for 1 h at RT in 330 mL/L dimethyl sulfoxide in 20 mmol/L 4-morpholineethanesulfonic acid (pH 6.1) with 1.1 mmol/L of both 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide to establish a sulfo-N-hydroxysulfosuccinimide ester intermediate. After several washes, 1 mg of activated phosphor particles was mixed with 10–200 µg of antibody in a final volume of 1 mL of dimethyl sulfoxide–4-morpholineethanesulfonic acid buffer. The reaction was allowed to proceed for 2 h at RT and stopped with 30 µL glycine (2 mol/L, pH 11). After several washes, phosphor conjugates (1 g/L) were stored at 4 °C in 50 mmol/L glycine, 0.3 mL/L Triton X-100, and 1 g/L NaN3, pH 8.0.
target amplification and probe production
To amplify the 452-bp L1 DNA fragment from cervical carcinoma, we performed PCR on 10 ng of genomic DNA using the primer combination LF65-LF66 (Table 1
). Primer combination LF65-LF66 is the nondegenerated form (100% similarity with HPV16-L1 (17) of primer set MY09-MY11 (16), a primer set also used for general HPV detection under low stringency PCR conditions. PCR stringency conditions used in the experiments described here make primer set LF65-LF66 HPV16 specific. When necessary, we introduced biotin and DIG end-labels using the LF65 5'-biotin equivalent (LF99) and the LF66 5'-DIG equivalent (LF92), respectively (Table 1
).
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Genomic DNA from CaSki and 143B cells was used to develop and evaluate the described test systems. The 452-bp HPV16 L1 fragment (if present) was amplified as described above. When necessary, we introduced biotin and DIG end labels using the primer set LF99-LF92. A 560-bp E6–E7, negative-control fragment was amplified from CaSki DNA with primer combination LF75-LF76 (Table 1
).
L1-specific probes were amplified from CaSki DNA with the primer combination LF65-LF69 and LF87-LF90, leading to a 109-bp biotin end-labeled and a 296-bp DIG end-labeled DNA fragment, respectively. Probes were excised from ethidium bromide-stained agarose gels and further purified with the QIAquick Gel Extraction Kit (Qiagen Inc.).
PCR reactions were performed as follows: an initial 5-min denaturation step at 95 °C, followed by 25 (prescreening) or 30 cycles (hybridization-based test) of 30 s at 95 °C, 45 s at 55 °C, and 45 s at 72 °C; sample volume was 100 µL; 10 pmol of primer (0.1 µmol/L final concentration) and 2.5 U of Taq polymerase (Promega Benelux b.v.) were used per 100 µL of reaction; the Taq polymerase was added at the end of the initial denaturation step (hot-start method) to reduce primer-dimer (PD) formation, and amplification was performed in a PTC-200 thermocycler (MJ Research Inc.).
Unless indicated otherwise, we purified PCR samples using the QIAquick PCR Purification Kit (Qiagen Inc.) to allow accurate A260 measurements for DNA determination. For the described UPT LF assays, Qiagen purification can be omitted without any loss in performance.
prescreening assay
Generally, 2 µL of diluted or undiluted DIG-biotin-labeled PCR sample was added to 18 µL of flow buffer (10 mmol/L HEPES, pH 7.2; 135 mmol/L NaCl, 10 g/L bovine serum albumin; and 50 mL/L Tween). This material was added to 200 µL of phosphor conjugate (containing 200 ng of phosphor particles) and analyzed on two duplicate LF strips as described below.
hybridization-based assay (verification test)
All hybridizations were performed in 2x standard saline citrate (18) and 10 mL/L Tween 20 in a background of 1 µg/µL fish sperm DNA (single-stranded DNA, 120–3000 nucleotides in length; Roche Diagnostics Netherlands) at 56 °C for 1 h in a thermocycler (PTC-200) with heated lid. To perform the hybridization, target DNA (2 µL of the undiluted or diluted PCR sample) and both probes (30 fmol of 1.5 fmol/µL; 2 and 6 ng of the biotin and DIG probes, respectively) were mixed with 20 µg of fish-sperm DNA (final concentration, 1 µg/µL) and adjusted to a final volume of 20 µL. The samples were denatured for 5 min at 95 °C and then rapidly cooled to the hybridization temperature of 56 °C. A hybridization time of 1 h was generally used. Denaturation and hybridization were performed in a thermocycler (PTC-200) with a heated lid to avoid evaporation. After hybridization, samples were immediately mixed with 200 µL of phosphor conjugate (containing 100 ng of phosphor particles) and analyzed on two duplicate strips by LF analysis.
LF and strip analysis
After the addition of the phosphor conjugate, all samples were split in two and flowed over duplicate strips (same batch). In general, to 20 µL of sample was added 200 µL of flow buffer (10 mmol/L HEPES (pH 7.2), 135 mmol/L NaCl, 10 g/L bovine serum albumin, 5 mL/L Tween) with 10 µg of fish-sperm DNA and 100 ng of MDIGPHOS conjugate. For the hybridization-based assays, fish sperm DNA was added to the hybridization mixture instead of to the flow buffer. The sample and flow buffer were mixed and pipetted into microtiter wells in which LF strips were placed to perform a vertical flow. A standard flow time of 30 min was applied before the strips were scanned.
A Packard FluoroCountTM microtiter-plate reader was adapted with an infrared laser (980 nm) and modified to scan LF strips. After IR excitation, phosphor particles emitted green light that we detected with a 550 nm filter. The strips were scanned and luminescence was measured, compiled, and displayed as relative fluorescence units in a two-dimensional plot with respect to the position on the strip. The software used for analysis included algorithms for background correction and peak-area determination. Further calculations were performed with Microsoft Excel. Peak areas of the avidin target-capture lines were normalized to the peak area of each individual goat anti-mouse IgG antibody control line and presented as histograms.
Results
We describe here the first application of UPT reporters in LF systems to detect specific nucleic acids. The test system developed is divided into a rapid and sensitive prescreening test (Fig. 2A
) and a more specific hybridization-based confirmatory test (Fig. 3A
).
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evaluation of the prescreening: specificity of the avidin capture
The first aspects that were investigated concern the condition under which DNA molecules, indirectly labeled with phosphor particles, can be flowed and specifically captured by avidin. With primer pair LF65-LF66 and the corresponding biotinylated (LF99) and DIG-labeled (LF92) primers, the 452-bp fragment of the L1 gene of HPV16 was amplified from genomic DNA of CaSki cells. Primer combinations were chosen such that fragments were obtained containing either a single DIG or single biotin label, both labels, or no label at all. To avoid interference of possible PCR artifacts, the 452-bp fragments were excised from the gel and further purified. From each sample, 1 ng of PCR product was applied to the MDIGPHOS conjugate and flowed over a LF strip. The results are presented in Fig. 2B
. Only the fragment double labeled with DIG and biotin showed a substantial signal at the target-capture line. The highest nonspecific signal was obtained with the fragment containing a DIG hapten only, although this signal was still a factor of 10 lower than the specific signal. With lower or higher amounts of PCR product (1 pg or 10 ng, respectively), the nonspecific binding as compared with specific capture was never >20% (results not shown).
evaluation of the prescreening: pcr artifacts, PDs
With the avidin-capture test described above, one cannot differentiate between specific PCR products and PCR artifacts, such as PDs. PCR artifacts that contain both haptens will react similarly to the specific products and thus generate a false signal at the target-capture line. We investigated PD formation by analyzing DIG-biotin-labeled PCR samples taken after 10, 25, and 40 cycles (Fig. 2C
). The results clearly indicated that after 40 cycles, PD formation becomes a problem. Up to 25 cycles, PD formation does not interfere with the test result of the prescreening. Note, furthermore, that saturation rarely occurs in a 25-cycle PCR, indicating that quantification of the amount of target molecules present in the original test sample is possible. However, in the context of HPV screening, quantification is less relevant because the primary goal is to specifically detect positive samples.
evaluation of the prescreening: detection limit and amount of genomic dna required
We determined the minimum amount of original genomic DNA needed for PCR amplification of HPV16 sequences to cause a positive UPT LF test. As mentioned above, with the use of gel-purified, DIG-biotin-labeled PCR fragments with UPT LF tests, the presence of <1 pg of a 452-bp product (3 attamol) could be demonstrated. To investigate the minimum amount of original genomic DNA needed, a dilution series of genomic CaSki DNA was amplified in a 25-cycle PCR, and the resulting samples were analyzed with UPT LF. The results are presented in Fig. 2D
. Under the described PCR conditions, 100 pg is the minimum amount of genomic CaSki DNA needed in a PCR to produce a positive signal with UPT LF analysis. This translates to the equivalent of approximately eight (tetraploid) CaSki cells with 200–800 copies of integrated HPV16 per cell. From these results, we concluded that a minimum of 10 ng of genomic DNA is required for the prescreening test proposed here. This implies a detection of 2000 (diploid) cells with a single copy of HPV. In other words, any DNA sample containing >2000 copies of HPV per 10 ng of total DNA may be detected.
evaluation of the hybridization-based assay: probe concentration
The optimal concentrations of the 109-bp biotin-labeled and 296-bp DIG-labeled probes (Fig. 3A
) were determined by performing hybridizations with the 452-bp L1 fragment amplified from genomic CaSki DNA. Probe concentrations were tested in the range of 0.15–15 fmol/µL. The molar ratio between biotin and DIG probe varied among 3:1, 1:1, and 1:3. The result obtained with the optimum probe concentration of 1.5 fmol/µL in a 1:1 ratio is shown in Fig. 3B
. Specific signals obtained with the CaSki PCR sample were 120- and 129-fold higher than the nonspecific signals with 143B and water PCR samples, respectively. Higher probe concentrations led to lower signals because of the competitive interaction between free probe and avidin- and MDIG-binding sites. Significant probe-probe interactions were not observed. Lower concentrations led to lower signals as a consequence of probe limitation.
evaluation of the hybridization-based assay: nonspecific hybridization
Nonspecific hybridization can lead to false-positive results in cases where samples are analyzed by LF. The probability of nonspecific hybridization was investigated comparing the UPT LF signal obtained with the 452-bp LF65-LF66 CaSki target (part of the L1 gene) and the signal obtained with a nonspecific target. The nonspecific target used was a 560-bp HPV16 fragment encoding the largest parts of genes E6–E7 (LF75-LF76; Table 1
). Hybridizations were performed with 10 ng and 100 pg of the L1 and E6–E7 PCR products. In the 100 pg situation, the probes are in 20-fold excess, a situation close to circumstances used in conventional hybridization reactions. In both situations, the specific signal is well above the nonspecific signal. In the 100 pg situation a nonspecific signal was not even observed. It is therefore concluded that the specificity of the hybridization reaction is ensured for the test conditions described.
evaluation of the hybridization-based assay: sensitivity and detection limit
For the chosen model (HPV16 detection), the detection limit of the hybridization-based verification test is of secondary importance. However, the hybridization-based assay may also be applied as a stand-alone test for other diagnostic applications when the detection does play a key role. DNA dilution series (10 ng to 1 pg) of the 452-bp L1 and 560-bp E6–E7 fragments were therefore analyzed with the hybridization-based assay. Signals obtained with nonspecific E6–E7 target were corrected for (subtracted from) signals obtained with equal amounts of specific L1 target. All experiments performed allowed convenient and reproducible detection of 10 pg (30 attamol) of target (Fig. 3D
). In some tests, detection of 1 pg was shown feasible. In comparison, when using LF strips with gold detection, we never observed positive signals from the 1-ng target.
prescreening of uterine cervix carcinoma samples
The results of the prescreening analysis of 10 cervical carcinoma and control samples are presented in Fig. 4A
. The 100% and 200% (defined as twofold the 100% value) cutoff values were determined as the signals plus the SD obtained with the largest values of the two negative controls, 143B DNA and water. All samples between the 100% cutoff value (0.14) and 200% cutoff value (0.28) that required verification with the hybridization-based verification assay were considered indeterminant. Carcinoma samples 1, 2, 3, 5, and 6 were well above the 200% cutoff value and were therefore classified HPV16 positive. Carcinoma samples 4 and 9 fell between the 100% cutoff and 200% cutoff values and were therefore marked indeterminant. Carcinoma samples 7, 8, and 10 were below the cutoff value and were therefore classified HPV16 negative. The prescreening identified five HPV16 positives and two potential indeterminants that needed verification by the hybridization-based assay. In comparison, with equal amounts of PCR product on LF strips with gold detection, none of the carcinoma samples led to a positive signal.
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hybridization-based analysis of uterine carcinoma samples
All carcinoma samples (not only samples 4 and 9) were then analyzed with the hybridization-based assay (Fig. 4B
). Cutoff values were determined as described for the prescreening assay. In this particular experiment, both negative controls did not generate any signal. Therefore, carcinoma samples 1–6 could all be marked as HPV16 positive, and carcinoma samples 7–10 could all be marked HPV16 negative. These results matched 100% with previous characterization of the carcinoma.
Discussion
LF devices (LFDs) are increasingly used for onsite testing of analytes. Applications range from home pregnancy tests to detection of drugs by federal authorities (3). Diagnostic companies have expressed increased interest in LFDs because of their enormous diagnostic potential in the area of infectious diseases. There are three important limitations of the current LFDs that hamper their diagnostic use.
One limitation is that the commonly used labels, e.g., latex particles and colloidal gold, lack the sensitivity needed for many of the new diagnostic applications (1). Another limitation is that LFDs mostly provide qualitative information, whereas in diagnosis, the ability to quantify is often a prerequisite. Furthermore, current tests are mostly based on the specific interaction of immunoreagents with analytes. Detection of haptenized amplified DNA can obviously be accomplished with the use of immuno-based, LF assays. To date, however, reports describing the identification of disease-related, defined nucleic acid sequences on the basis of hybridization and LF are scant.
We demonstrate in this report substantial progress toward successfully addressing each of the three problems listed above. A new reporter technology, UPT, is used as the assay probe reporter. In other applications such as immuno-LF assays (9)(10), as well as with DNA microarraying (11), UPT particles have been very sensitive.
In this report, it is shown that standard immuno-LF assays can be modified to detect specific DNA sequences. Haptenized and indirectly labeled UPT DNA (labeled with antibodies conjugated to UPT particles) flows through nitrocellulose and can be specifically captured and measured, a very basic but important conclusion. More interesting is the possibility of flowing and capturing DNA targets that were initially hybridized to two gene-specific DNA probes, one containing a hapten for capture and one that is indirectly attached to UPT particles for detection.
On the basis of these fundamental findings, we developed a strategy to specifically detect HPV16 DNA targets. We chose HPV as a model because of the large amount of existing knowledge on HPV and its large diagnostic potential. More specifically, type 16 was chosen because it is considered a sensitive screening marker for the detection of cervical cancer.
The strategy to detect HPV16 infection combines a rapid and sensitive prescreening with a genotype-specific, hybridization-based verification assay. Both assays include an initial PCR step in which the target (part of the L1 gene from HPV16) is amplified from genomic DNA. The prescreening uses only a limited amount of PCR cycles (up to 25) and is easily performed under standard conditions. The subsequent hybridization-based assay is a verification test of those samples that are classified positive or potentially positive by the prescreening test. The specificity of the total test is therefore not only based on the specificity of the PCR amplification but also on the consecutive hybridization steps requiring the specific hybridization of two probes. In addition, the hybridization-based assay does not suffer much from PCR artifacts (as e.g., PDs) when the PCR includes a high number (>30) of amplification cycles.
For the preliminary set of samples that were tested, we have shown that with the above strategy, the six HPV16-positive samples were correctly classified. The results matched 100% with previous characterization of the cervical carcinoma samples. Obviously, substantial testing of a large series of cervical cancer samples is needed to determine the best cutoff value for optimal specificity and sensitivity to allow accurate statements about the reliability of HPV16 testing with this approach.
In conclusion, the results of this model study show the large potential of the UPT LF-DNA assay format. The fact that the entire test is easily performed and evaluated under standard conditions with the use of a portable bench-top reader makes the technology well suited for onsite testing of a large assortment of pathogens and onsite diagnosis of the diseases involved.
Footnotes
1 Nonstandard abbreviations: LF, lateral flow; DIG, digoxigenin; IR, infrared; HPV, human papillomavirus; RT, room temperature; MDIGPHOS, mouse anti-DIG antibody conjugated to phosphor particles; LFD, LF device; and PD, primer-dimer. 
References
The following articles in journals at HighWire Press have cited this article:
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