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1 Department of Pharmaceutical Sciences;
2
Department of Clinical and Experimental Medicine, Division of Microbiology; and
3
Institute of Chemical Sciences, University of Bologna, 40126 Bologna, Italy.
aAddress correspondence to this author at: Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy. Fax 39-051-343398; e-mail roda{at}alma.unibo.it.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Seven different oligonucleotide probes, each specific for a high-risk HPV genotype, were separately immobilized in the subwells. Subsequently, a digoxigenin-labeled consensus PCR amplification product was added to the main well. The PCR product hybridized to the immobilized probe corresponding to its genotype and was subsequently detected by use of a peroxidase-labeled anti-digoxigenin antibody and chemiluminescence imaging with an ultrasensitive charge-coupled device camera. Results obtained for 50 cytologic samples were compared with those obtained with a conventional colorimetric PCR-ELISA.
Results: The method was specific and allowed detection of 50 genome copies of HPV 16, 18, 33, and 58, and 100 genome copies of HPV 31, 35, and 45. Intra- and interassay CVs for the method were 5.6% and 7.9%, respectively. All results obtained for clinical samples were confirmed by the conventional PCR-ELISA.
Conclusions: PCR-CLEIA allows rapid, single-tube simultaneous detection and typing of seven high-risk HPV DNAs with small reagent volumes. The principle appears applicable to the development of other single-tube panels of tests.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have developed a new microtiter plate format for simultaneous multianalyte single-tube detection. The plate contains 24 main wells, each divided into 7 subwells. Localization and quantification of the signal in each subwell requires a chemiluminescent detection system (10), an approach that also provides higher detectability and a wider dynamic range than colorimetric detection (11)(12)(13). Imaging of the microtiter plate allows simultaneous measurement of the chemiluminescent signal localized in each subwell. To explore its clinical potential, we have used the plate as a support for a newly developed PCR-chemiluminescent enzyme immunoassay (CLEIA) for simultaneous detection and typing of the seven most common high-risk HPV DNAs (HPV 16, 18, 31, 33, 35, 45, and 58). The assay is based on consensus PCR amplification of a conserved sequence of 30 HPV genital genotypes and on the typing of 7 high-risk HPVs by hybridization followed by a chemiluminescent immunoassay. We applied this strategy for rapid diagnosis of high-risk HPV infection to 50 cytologic specimens from women with suspected HPV infection and compared the results with those obtained by conventional PCR-ELISA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PCR was performed on a Hybaid PCR Express Thermal Cycler. Chemiluminescence imaging was performed on a Night Owl LB 981 luminograph (EG&G Berthold).
new microtiter plate format
The 24 x 7-well microtiter plate, which has been designed in our laboratory and manufactured by Termodesign s.r.l., has a conventional 86 x 128 mm frame (height, 8 mm) to take advantage of the available automation systems and robotics for reagent dispensing, incubation, and washing steps. The plate format consists of 24 main wells (diameter, 18 mm), each of which is divided into 7 subwells (diameter, 4 mm), as shown in Fig. 1
. The optimum volume required for assay performance is 200 µL for each main well, whereas the volume necessary for bioprobe immobilization is 10 µL for each subwell. Four different versions in black or white polystyrene, with glossy or matt finishes, were produced as prototypes.
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samples for analytical validation (cell lines, plasmids, and reference samples)
Analytical validation of PCR-CLEIA was performed with two HPV-positive cervical carcinoma cell lines as positive controls: SiHa, which contains 1–2 genome copies of HPV 16 DNA/cell (ATCC no. HTB35), and HeLa, which contains 20–50 genome copies of HPV 18 DNA/cell (ATCC no. CCL-2) (14). The HPV-negative erythroblastoid human cell line TF-1 was used as a negative control. Seven plasmids containing the L1 region of HPV 16, 18, 31, 33, 35, 45, and 58, respectively, were used, along with two HPV-positive (HPV 16 and 31 genotype) and two HPV-negative reference cervical specimens.
clinical samples
For clinical validation of the assay, we evaluated a total of 50 cytologic specimens for the presence of HPV DNA. The 50 specimens were collected by a Dracon-tipped swab from the endo-ectocervix of 50 women with clinically suspected HPV infection. Cytologic specimens, collected in phosphate-buffered saline solution (10 mmol/L Na2HPO4 · H2O, 1 mmol/L KH2PO4, 0.1 mol/L NaCl, 2 mmol/L KCl, pH 7.4), were centrifuged, and the cells were counted and then divided into aliquots containing 1 x 105 cells and centrifuged again. The pellets were conserved at -70 °C until use.
All enrolled patients received a briefing on the purpose and nature of the study before providing written, informed consent. The local Medical Ethics Committee approved the study protocol and written informed consent form.
samples preparation
Aliquots containing 1 x 105 cells of the SiHa, HeLa, and TF-1 cell lines, from HPV-positive and -negative reference samples and clinical samples, were protease-digested by incubation at 55 °C for 2 h in 200 µL of digestion buffer [50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 0.5 mL/L Tween 20, and 400 mg/L proteinase K], followed by heat inactivation at 95 °C for 10 min. Plasmids were extracted by use of a DNA Matrix (Plasmid Midi Kit; Qiagen), and HPV genome concentrations, expressed as genome copies/µL, were determined by spectrophotometer. Serial 10-fold dilutions up to 10-6 of HeLa or SiHa cell lysates (5 x 102 cells/µL) were prepared; serial 10-fold dilutions of each of the seven plasmids were also prepared, containing 105–10-1 HPV genome copies/µL.
primers and probes
Consensus primer pairs MY09 and MY11 (15), able to detect >30 different genital HPV types within the L1 open reading frame, were used in PCR amplification.
Oligonucleotide probes MY14, WD74, WD128, MY59, MYB117, MY70, and MYB179 (5)(16), which are specific for the L1 PCR products of HPV 16, 18, 31, 33, 35, 45, and 58, respectively, were used for hybridization reactions.
pcr-cleia method
DNA amplification and labeling.
DNA amplification and labeling were performed in a 50-µL PCR volume containing 50 mM KCl; 10 mM Tris-HCl (pH 8.2); 4 mM MgCl2; 0.1 mM each of dATP, dGTP, and dCTP; 0.095 mM dTTP; 0.005 mM digoxigenin-11-dUTP; 0.1 µM each primer; 2.5 U of Taq DNA polymerase; and 10 µL of each sample (reference or clinical), cell lysate dilution, or plasmid dilution. After an initial denaturation step at 95 °C for 5 min, 40 cycles were performed, each consisting of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min; the final extension step was at 72 °C for 5 min.
Hybridization and detection reaction.
For each main well, seven oligoprobes specific for HPV 16, 18, 31, 33, 35, 45, and 58, respectively, were adsorbed to individual internal subwells. In particular, 10 pmol of each oligoprobe was diluted in 10 µL of 1.5 mol/L NaCl and dispensed in each individual subwell. After overnight incubation at 37 °C, 400 µL of blocking solution (10 g/L bovine serum albumin in phosphate-buffered saline solution) was added to each main well, and the plate was incubated at 37 °C for 60 min. The coated plates were most often used immediately; for longer storage, they were dried under reduced pressure and stored at 4 °C in sealed plastic bags together with a 5-g package of silica gel desiccant (Sigma) for up to 4 weeks.
A prehybridization step was performed by adding 400 µL of DNA calf thymus (100 mg/L in hybridization buffer) to each main well and incubating at 55 °C for 60 min. Subsequently, 20 µL of amplified product was added to 20 µL of denaturation buffer (100 mmol/L NaOH, 1 mL/L Tween 20) and incubated a 25 °C for 5 min. After the addition of 160 µL of hybridization buffer, the mixture was dispensed in the main well. The hybridization reaction was performed at 55 °C for 90 min. After five rinses with 400 µL of wash buffer, 200 µL of POD-conjugated anti-digoxigenin (20 U/L in conjugate buffer) was added to each main well and incubated at 25 °C for 30 min. After five more rinses in wash buffer and addition of 200 µL of the substrate SuperSignal ELISA Femto to each main well, the chemiluminescent signal was measured. The chemiluminescence intensity of each internal subwell was measured, integrating the photon emission over its entire area. The results were reported in photons · pixel-1 · s-1 for each subwell.
Because the values obtained for the TF-1 negative control were in the range of values obtained for the reference HPV-negative specimens (P <0.05), the cutoff of the reaction was determined for each probe as two times the mean value of HPV-negative TF-1 cell lysate. Test samples with values within ±20% of the cutoff value were retested for confirmation.
conventional pcr-elisa method
A 10-µL volume of each digested sample was processed by a conventional PCR-ELISA method as described previously (17)(18). In brief, after DNA amplification and labeling as described above, amplified products, which were digoxigenin-labeled during amplification, were separately hybridized with type-specific biotinylated probes (for HPV 16, 18, 31, 33, 35, 45, and 58). Hybrids were captured on streptavidin-coated 96-well microtiter plates and detected by means of a POD-labeled anti-digoxigenin antibody and by colorimetric detection with ABTS substrate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10-fold greater than the instrumental background, which disappeared altogether after 10-min incubation in the dark. No significant difference was observed between matt or glossy plates of the same color. Therefore, with white plates the chemiluminescence emission was subsequently measured after incubation for 10 min in the dark.
The four different microtiter surfaces were investigated to evaluate their capacity to adsorb nucleic acids and to provide optimal signal detectability. Three different concentrations (1, 10, and 100 pmol/subwell) of MY14 or WD74 probes were immobilized on each plate. Subsequently, PCR products obtained from 10 µL of serial 10-fold dilutions of HPV-positive SiHa or HeLa cell lysates (5 x102 cells/µL) were assayed with the immobilized specific oligoprobe. No cross-talk of the chemiluminescent signal among adjacent subwells was observed in any of the four prototypes, as shown in Fig. 1
. Thus, the signals emitted from different subwells were spatially resolved within the main well, enabling accurate individual measurements. Comparison of calibration curves obtained with the same oligoprobe concentration in the four different prototype plates revealed that plates with a matt finish provided lower limits of detection and higher sensitivities; no significant color-related difference was found. Because white plates provided higher chemiluminescence emission signals, the matt white format was selected for use in all subsequent experiments. Comparison of curves obtained with various immobilized oligoprobe concentrations on these plates revealed that the calibration curves with the lowest limit of detection and the highest sensitivity (slope of the curve) were obtained when 10 pmol/subwell of each oligoprobe was used, with no further improvement being obtained at higher oligoprobe concentrations. Therefore, we used 10 pmol/subwell for each oligoprobe in all subsequent experiments.
optimization of pcr-cleia method
Limit of detection.
Limits of detection of the newly developed PCR-CLEIA method, using matt white plates and 10 pmol/subwell each oligoprobe, were determined with SiHa and HeLa HPV-positive cell lines as well as seven plasmids containing the L1 regions of HPV 16, 18, 31, 33, 35, 45, and 58, respectively. Serial 10-fold dilutions up to 10-6 of SiHa or HeLa cell lysates (5 x102 cells/µL) and of each of the seven plasmids (105 to 10-1 HPV genome copies/µL) were PCR amplified. The amplification products were hybridized with the immobilized type-specific HPV probes. The results are shown in Fig. 2
. The limit of detection for each amplicon was determined as the chemiluminescent signal 20% above cutoff. The PCR-CLEIA method allowed detection of 50 genome copies of HPV 16, 18, 33, and 58, and 100 genome copies of HPV 31, 35, and 45.
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Specificity.
Serial 10-fold dilutions up to 10-6 of SiHa, HeLa, and TF-1 cell lysates (5 x 102 cells/µL), serial 10-fold dilutions of the seven plasmids (105 to 10-1 HPV genome copies/µL), and the reference samples were PCR amplified and hybridized with all HPV probes. A positive signal was detected when PCR-amplified SiHa and HeLa cell lysate dilutions, plasmids, and reference HPV-positive samples were hybridized with the specific oligoprobes. A signal within the background range (negative signal) was detected when PCR-amplified TF-1 cell lysate and reference HPV-negative samples were hybridized with immobilized oligoprobes. Moreover, no cross-reaction was observed when amplified products from SiHa and HeLa cell lysates, plasmids, and reference HPV-positive samples were hybridized with non-type-specific probes (data not shown).
Precision.
To investigate the reproducibility of the PCR-CLEIA method, we amplified two HPV-positive reference samples (HPV 16 and 31) and two HPV-negative reference samples. Amplification products were then assayed with type-specific immobilized oligoprobes in triplicate in three independent assays. The average intraplate variation (CV) was 5.6%, whereas the interplate CV was 7.9%.
Robustness of the method.
The stability of the oligoprobe-coated microplates was investigated as follows. After internal subwells were coated with specific oligoprobes and incubated with blocking solution, the plates were dried under reduced pressure and stored at 4 °C in sealed plastic bags, as reported above. The plates were used after storage for 0, 1, and 4 weeks. In particular, after prehybridization with calf thymus DNA, serial 10-fold dilutions of two plasmids (containing the HPV 16 and 18 L1 regions) ranging from 105 to 10-1 HPV genome copies/µL were PCR-amplified and hybridized with type-specific HPV probes. Storage of coated microplates under reduced pressure at 4 °C for up to 4 weeks did not significantly alter the performance of the assay in terms of limit of detection and sensitivity (data not shown).
clinical validation
Fifty clinical samples were analyzed with both the newly developed PCR-CLEIA method and the conventional PCR-ELISA method (17)(18). Amplification product from a TF-1 cell lysate (5 x 102 cells/µL) was used in each polystyrene microtiter plate as a negative control, and the cutoff was calculated for each probe as two times the mean value of a negative TF-1. Concordant results were obtained with the two methods: 22 samples were concordantly negative and 28 concordantly positive (9 for HPV 16 DNA, 3 for HPV 18 DNA, 2 for HPV 31 DNA, 4 for HPV 33 DNA, 1 for HPV 35 DNA, 2 for HPV 45 DNA, 2 for HPV 58 DNA, 2 for HPV 16 and 18 DNA, 2 for HPV 16 and 33 DNA, 1 for HPV 31 and 33 DNA); all the HPV genotypes detected by the developed method corresponded to those obtained by the conventional PCR-ELISA method, even in cases of samples positive for two different HPV genotypes. As shown in Fig. 1
, no cross-reaction was observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epidemiologic and molecular biology studies have shown a causal relationship between HPV infection and cervical neoplasia, with high, intermediate, or low oncogenic risk associated with different HPV genotypes (20)(21). Therefore, genotyping of any detected HPV infection is important to assess the degree of oncogenic risk. Our newly developed PCR-CLEIA method is based on consensus PCR amplification of a conserved sequence of 30 HPV genital genotypes and on the typing of 7 high-risk HPVs in one well by hybridization followed by a chemiluminescent immunoassay.
The method involves initial immobilization of the seven probes in their respective subwells. We found that the coated plates can then be stored at 4 °C for at least 4 weeks in plastic bags sealed under reduced pressure without any impairment of sensitivity. After the immobilization step, all samples and reagents are directly dispensed to each main well. This greatly simplifies the multianalyte detection procedure and should reduce the risk of operator errors.
We have previously developed a sensitive chemiluminescent in situ hybridization assay for the detection of HPV genomes in biopsy specimens (22). In the present work, localization and quantification of POD activity within each subwell was performed by use of a similar chemiluminescence measurement system. Imaging of the microtiter plate allowed accurate simultaneous measurement of the signal generated by each of the 168 subwells. The enhanced luminol-based chemiluminescent substrate is characterized by glow-type emission kinetics, which permit easy handling and standardization of the experimental conditions. In the presence of an excess of substrate, the steady-state light emission, which occurs after 2–3 min and is maintained for at least 15 min, is proportional to the activity of the POD enzyme present in the subwell. The intensity of the steady-state chemiluminescent signal in each subwell can easily be quantified because of the topographic separation of the subwells, which are physically separated by walls, and the focusing optics and resolution offered by charge-coupled device light acquisition. This prevents cross-talk phenomena, which can be observed in microarray systems (19). The recorded intensity is then correlated to the amount of hybridized DNA. No cross-talk was observed among adjacent subwells, despite the use of a white finish, which could conceivably have presented this risk, because of the reflection of emitted photons by the surfaces of the well. Some commercially available systems allow multianalyte detection in a microtiter plate format and are based on the presence of different electrode surfaces within each well, separated by a dielectric layer. Detection is performed by electrochemiluminescence [Meso Scale Diagnostics LLC; Ref. (23)]. However, these systems are not entirely satisfactory for quantification purposes because a degree of cross-talk among adjacent spots can be observed.
Although photomultiplier tube-based instruments provide the best analytical performance in terms of light detection, recent improvements in slow-scan cooled charge-coupled device technology allow comparable performance (11). Because the plate has a conventional 86- x 128-mm frame, the measurement could theoretically be performed in a microtiter plate luminometer that allows custom programming of the xy position of the wells to be measured. However, the sequential reading of all 168 subwells could take as long as 5–10 min (depending on the signal integration time required for each well), whereas an imaging device allows instant, simultaneous measurement. Moreover, in the case of sequential measurements, one would have to check the constancy of the light output over the entire period of measurement, especially for high-emission subwells (12).
Use of an imaging detection system allowed accurate evaluation of the spatial immobilization of the oligoprobe within each subwell. For quantification purposes, we integrated the chemiluminescent emission over the entire subwell area. Nevertheless, it is worth noting that the use of an imaging detection system enabled us to observe the spatial distribution of the signal within the subwell area, which showed a good degree of homogeneity of oligoprobe immobilization (data not shown).
We used cell lines, plasmids, and reference samples to explore the analytical performance of the newly developed PCR-CLEIA method. The detection limit of PCR-CLEIA was 50 genome copies of HPV 16, 18, 33, and 58 DNA, and 100 genome copies of HPV 31, 35, and 45 DNA. This is similar to the sensitivity of conventional PCR-ELISA (17)(18). The different limits of detection observed for the various HPV genotypes are mainly attributable to the different amplification efficiencies observed when consensus PCR amplification is used (24). Furthermore, the method was specific and precise (intraplate CV, 5.6%; interplate CV, 7.9%). We also explored the diagnostic potential of the method on clinical samples. PCR-CLEIA was demonstrated to be a specific and reliable assay for the identification of different HPV DNAs. The viral DNA found with our assay corresponded with the expected HPV genotype in all the positive samples examined, and no HPV DNA was detectable in any of the negative samples.
Although we limited our analysis of the sensitivity and specificity of the PCR-CLEIA method to seven high-risk HPV genotypes, the approach could be extended to other genotypes for which specific oligoprobes are available. The reduced sample volume enables seven genotypes to be assayed in just 20 µL of PCR product, whereas the PCR-ELISA method requires 35 µL. Because two 20-µL aliquots can readily be taken from 50 µL of PCR amplification product, this allows typing of at least 14 HPV genotypes, compared with a maximum of 9–10 with PCR-ELISA. Furthermore, the single-tube method reduces the amounts of reagents and sample volumes required, thus reducing time and costs with respect to PCR-ELISA.
In conclusion, this newly developed multianalyte PCR-CLEIA method allows straightforward and economic detection and typing of seven high-risk HPV DNAs with the required sensitivity and specificity. PCR-CLEIA provides major potential savings in costs with respect to both PCR-ELISA and the most recent microarray or microchip methods. In the future, the novel microtiter plate format may also permit development of multianalyte assays for other pathologic conditions, thereby providing easily performed panels of tests for specific diseases. The tests could involve detection of cDNA, RNA, antigens, antibodies, haptens, and receptors by separate immobilization of the specific capture probes within each main well. In addition, the layout of the plate can be custom designed by dividing each main well in the desired number of subwells to allow performance of the number of tests necessary for the diagnosis of a particular disease. Such simultaneous quantification of independent biomarkers could have considerable diagnostic potential in various clinical situations.
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Acknowledgments |
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Footnotes |
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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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  M. Magliulo, B. Roda, A. Zattoni, E. Michelini, M. Luciani, R. Lelli, P. Reschiglian, and A. Roda An innovative, flow-assisted, noncompetitive chemiluminescent immunoassay for the detection of pathogenic bacteria. Clin. Chem., November 1, 2006; 52(11): 2151 - 2155. [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or HeLa (
) cells contain 5 x 102 cells/µL. (B), plasmids contain the L1 regions of HPV 16 (
), 18 (
), 33 (
), 35 (
), 45 (•), or 58 (
). The cutoff of the reaction, determined as two times the mean value of a negative control, TF-1, is indicated by the dotted line. Bars, SD.