Medical Device Link.

Originally Published IVD Technology March 2002

Molecular Diagnostics

Fluorescent markers for high-throughput DNA screening applications

A primer-based fluorescence detection system offers an alternative especially suited for automating homogenous DNA assays.

Sandra K. Randall, Jay Ji, Bita Nakhai, Nate Lawrence, and Mark Manak

A depiction of high-throughput PCR screening for blood-borne pathogens utilizing the Amplifluor detection system.

Most such tests employ some method of amplification in order to enable the detection of the smallest possible quantities of target DNA in clinical samples. Common amplification strategies include target amplification (nucleic acid sequence-based amplification, strand displacement amplification, PCR), probe-based amplification (cascade rolling-circle amplification), and signal amplification (branched DNA, Digene hybrid capture assay).^1–5

Homogenous assay methods offer the advantage that they do not require the operator to perform manual separation of the amplified target by means of gel electrophoresis or other methods. Once setup is complete, target detection can be accomplished without additional manipulation of the sample.

A major advance for PCR-based tests has been the development of homogenous assays using fluorescence detection. Such assays facilitate high throughput by monitoring the accumulation of fluorescence in a closed tube. Once the sample extract and reagents are combined, the tube is sealed and does not need to be opened again. This method minimizes the likelihood of false-positive results due to carryover contamination of the sample (a notable shortcoming of many nucleic acid amplification–based detection systems), facilitates sample tracking, and significantly reduces hands-on processing time.

Fluorescent Alternatives

PCR-based fluorescent homogenous assays can be monitored using either a labeled hybridization probe (TaqMan, Molecular Beacons) or a labeled PCR primer (Amplifluor).^6–10 When used with an instrument capable of measuring fluorescence accumulation in real time, each of these methods offers high-sensitivity target detection and quantitative measurements over a wide dynamic range. In real-time analysis, a PCR reaction is performed for a set number of cycles, and the amount of fluorescence is monitored during each cycle. The accumulation of fluorescence correlates to the amount of PCR amplicon generated during thermocycling. When a closed-tube system is employed, detection of the fluorescent signal generated during the PCR reaction does not require any labor-intensive postamplification processing.

The TaqMan assay detects the accumulation of a specific PCR product by hybridization and cleavage of a dual-labeled fluorogenic probe during the PCR extension step.^6,7 The fluorogenic probe consists of an oligonucleotide labeled with both a fluorescent reporter dye (5') and a quencher dye (3'). During PCR, probe hybridized to the segment being amplified is cleaved by the 5'-exonuclease activity of DNA polymerase. Cleavage of the probe generates an increase in the fluorescent intensity of the reporter dye.

Figure 1. Operation of the Amplifluor detection system. During the first PCR cycle, Amplifluor primer 1 anneals to the specific DNA target strand and is extended by Taq polymerase. During the second PCR cycle, Amplifluor primer 1 extension product serves as a template for primer 2 (the reverse primer). As primer 2 is extended by Taq polymerase, the Amplifluor hairpin primer is unfolded and a fluorescent signal is generated. Fluorescent signal generated during subsequent PCR cycles increases proportionally to the amount of amplified product. (click to enlarge)

Molecular Beacons are also oligonucleotide probes that hybridize to the amplicons generated during the PCR reaction.⁸ In this case, a donor fluorophore and a quencher moiety are located at the 5' and 3' ends of a hybridization probe which is designed to form a hairpin structure. When the beacon is in the hairpin conformation, fluorescence of the donor is quenched during excitation. During the PCR reaction, the molecular beacon probe hybridizes to the specific PCR product, resulting in the physical separation of the donor from the quencher and thereby permitting the donor fluorophore to fluoresce when excited at a specific wavelength.

Unlike these hybridization probes, the Amplifluor detection system works by incorporating energy transfer-labeled hairpin primers into the PCR amplification products (see Figure 1).⁹ Amplifluor primers contain target-specific sequences on their 3' ends and hairpin structures on their 5' ends, with donor and acceptor moieties located in close proximity on the hairpin stem. When the primer is free in solution, the hairpin structure brings the fluorophore and the quencher (4-[4'-dimethylamino-phenylazo] sulfonic acid; DABSYL) into close proximity with one another, permitting very efficient quenching of the donor fluorescence.

Upon incorporation into double-stranded amplification products, the hairpin structure of the Amplifluor primer is displaced. Within the double-stranded product, the distance between the donor fluorophore and the DABSYL acceptor is generally greater than 70Å, and emission from the fluorophore is detected following excitation. The increase in the fluorescent signal correlates directly to the accumulation of target-specific amplicons.

High-throughput screening methods can be made more efficient and cost-effective by replacing real-time analysis with endpoint analysis. In endpoint analysis, amplification is performed in standard thermocyclers, and detection of the fluorescent PCR-reaction signal is performed at the completion of a set number of amplification cycles. Quantitation of the signal can be achieved by using a fluorescence plate reader with the option for quantitative real-time analysis. Such a setup can significantly reduce total costs of the system by eliminating the need for screening by expensive real-time instruments that restrict sample throughput. In a typical PCR-lab arrangement using 96-well microplates, more than 10 thermocyclers can be coupled with a single fluorescence plate reader, making it possible to perform endpoint analysis of much greater numbers of samples.

However, researchers attempting to use hybridization-based fluorescence detection methods in a setup for endpoint-analysis can encounter difficulties. It has been observed that such methods offer a relatively low signal-to-noise ratio, which may be a result of either inefficient quenching (noise) or a low fluorescent signal. A primer-based detection system offers a solution to these difficulties because the hairpin structure of the primers provides more-efficient and consistent quenching of background fluorescence while incorporation of the fluorophore into the primer maintains a high fluorescent signal. This report presents a potential high-throughput assay under development for viral target screening using PCR amplification coupled with the Amplifluor detection system and an automated sample-processing method.

Viral Target Screening and Quantitation

Hepatitis B virus (HBV), the leading cause of liver disease, has infected about 0.5% of the U.S. population.^11–13 Diagnosis of HBV infection is based primarily on serological immunoassays for HBV antigens or antibodies and recently on HBV DNA.¹⁴ Serological assays, however, are not accurate quantitative indicators of current infection and cannot be reliably used to monitor viral replication. Studies have shown that nucleic acid amplification tests are earlier indicators of infection than serological immunoassays, offering an average reduction of the "window" period—the period during which HBV infection can first be detected—by 18.4 ± 10.0 days.¹⁵ Moreover, in the case of serologically negative mutants, DNA testing is the only reliable method for screening blood samples and monitoring HBV infection.^16,17

Figure 2. Real-time HBV target detection using Amplifluor primers. Serial dilutions of the HBV plasmid standard and extracted plasma samples were PCR amplified for 45 cycles. Amplification plot of plasmid standards; fluorescence was measured continuously at the annealing step (a). The threshold cycle (CT) for each plasmid standard was determined and plotted against the log (HBV target copy), generating a log linear standard curve (r2 = 0.99); the CT for each extracted plasma sample is plotted on the standard curve (b). (click to enlarge)

Three commercial assays are currently available for HBV quantitation, and several qualitative screening assays are under development. The most sensitive of the currently available assays is the Amplicor HBV Monitor (Roche Molecular Systems; Pleasanton, CA), a PCR-based colorimetric detection system with a quantitation range of from 400 to 40 million viral particles per ml. After viral lysis, the HBV s-gene fragment is PCR amplified with a biotin-labeled primer, hybridized with probes attached to a microwell plate, and detected by means of an avidin-conjugated antibody on the plate reader. Roche is currently developing a qualitative Ampli-Screen version for blood screening. The Digene Hybrid Capture HBV assay (Murex Diagnostics; Dartford, UK), and the Quantiplex HBV DNA assay (Bayer Diagnostics; Emeryville, CA) have quantitation ranges from 105 to 109 copies/ml. Chiron is collaborating with Gen-Probe Inc. to develop a more sensitive screening assay based on the transcription-mediated amplification (TMA) technology.¹⁸

All of the current HBV DNA assays—including those under development—require postamplification steps for signal detection. The quantitative assays (Roche Monitor, Digene Hybrid Capture, and Bayer Quantiplex) are coupled systems that require a large number of processing steps, expensive reagents, and extensive precautions to minimize the risk of PCR carryover. The screening assays currently under development (Roche Ampli-Screen and Gen-Probe/Chiron TMA) are also complex and expensive assays.

Homogenous and sensitive PCR-based fluorescence detection assays suitable for high-throughput viral screening are needed for screening blood and blood products. To be useful, a closed-tube PCR assay requires sensitivity below 100 viral copies/ml of blood. In addition, the screening system must be capable of reliably screening large numbers of samples—preferably in a cost-effective and automated format.

Using the Amplifluor technology, we have developed a system for HBV detection suitable for sensitive, high-throughput screening by endpoint analysis with the option for quantitative real-time analysis. In our system, viral DNA is first extracted using the Qiagen BioRobot 9604. Specific HBV targets are then amplified in a PCR reaction with Amplifluor primers in a 96-well microplate. Following amplification, as many as 20 microplates can be stacked on one fluorescence plate reader for automated endpoint analysis. The system couples automated sample processing and automated detection. Additional cost savings may be realized by screening sample pools representing many individual units of blood.

Assay Design

Figure 3. Endpoint screening of HBV plasma samples, showing the average RFU for each serial dilution of the Accurun 325 HBV-positive plasma sample analyzed in triplicate and three negative-plasma samples. Fluorescence was measured at the completion of PCR and is shown graphically above the predicted target concentrations for each sample. (click to enlarge)

Primer sets were carefully designed for efficient and specific amplification of the HBV s-gene fragment target sequence using Oligo 5 primer design software (Lifescience Software Resource; Long Lake, MN).¹⁹ PCR testing of the primer set was performed and validated by gel analysis to verify that target amplification occurred without PCR artifacts or mispriming. The Amplifluor HBV forward primer was synthesized by phosphoramidite chemistry to incorporate fluorescein at the 5' end and DABSYL at an internal T residue, and was then purified by high-pressure liquid chromatography. The HBV reverse primer was chemically synthesized by standard phosphoramidite chemistry.

For accurate quantitation, a plasmid vector containing the HBV s-gene was cloned, purified, and verified by DNA sequencing. Plasmid target DNA was quantitated by OD260 reading, and verified by fluorescent staining with Pico Green (Molecular Probes; Eugene, OR) and gel electrophoresis. Negative and HBV-positive plasma samples were extracted using the QIAamp 96 Blood BioRobot kit on the automated Qiagen BioRobot 9604 and amplified in 50-µl reactions.

Sensitivity and Linear Dynamic Range

Figure 4. Endpoint screening of normal donors. Plasma DNA from 18 normal blood donors was extracted manually and amplified using Amplifluor primers. Fluorescence was measured at the completion of PCR. Cutoff was set at 12,000 RFU. (click to enlarge)

Using the Qiagen BioRobot, serial dilutions (~102 to 105 copies/ml) of Accurun 325 HBV DNA-positive control series 600 (Boston Biomedica; West Bridgewater, MA) were extracted in triplicate with three negative-plasma samples. For both real-time and endpoint analysis, serial dilutions of plasmid standards were amplified for accurate quantitation and screening of plasma samples.

The amplification plot of the plasmid standard accurately detected 10 copies per assay (equivalent to 4 x 102 copies/ml) at 40 cycles (see Figure 2a). These results were based on extracting 200 ml of starting plasma and using one quarter of the sample in the amplification reaction. Further enhancement of sensitivity could be achieved by extracting a larger volume of plasma sample or by introducing a DNA concentration step prior to the amplification step.

Endpoint analysis after 45 cycles showed a similar trend, with plasma-sample sensitivity below 400 copies/ml of blood. This result suggests that a sensitivity of 10 target copies per assay would be suitable for high-throughput screening of blood products (see Figure 3). No amplification was detected in the three negative-plasma samples with either real-time or endpoint analysis.

Samples from 18 normal blood donors were also screened by endpoint analysis (see Figure 4). For this assay, a cutoff of 12,000 relative fluoresence units (RFU) for HBV detection was positioned between 102 and 103 copies/ml. All normal donors fell below the cutoff value.

Internal Control

Figure 5. Simultaneous amplification and detection of target and internal control with Amplifluor primers. During PCR, there are two different gene-specific Amplifluor primers for target detection. The excitation and emission wavelengths are sufficiently different to allow the simultaneous amplification and detection of HBV and internal control (IC) products in a single reaction vessel. The internal control is used as an indicator of DNA recovery and as a positive control for PCR amplification. (click to enlarge)

To limit false negatives and reproducibly detect plasma samples below 400 copies/ml of blood, an internal control (IC) was added to the sample prior to extraction. The IC makes it possible to monitor sample recovery and detect the presence of PCR inhibitors. Inclusion of an IC is especially important if sample concentration is required in order to achieve accurate and reproducible assay sensitivity of 100 target copies/ml.

The Amplifluor technology allows for the simultaneous amplification and detection of nucleic acids in a closed-tube format in the presence of an internal control for accurate real-time quantitation and endpoint formats (see Figure 5).²⁰ The IC contains a random sequence flanked with the IC reverse primer and an IC-specific Amplifluor primer labeled with sulforhodamine (SR). Initial studies have shown that assay sensitivity of less than 400 copies/ml can be maintained in the presence of 600 copies/ml of IC.

Conclusion

Homogenous and sensitive PCR-based fluorescence detection assays suitable for high-throughput viral detection are needed for screening blood and blood products. The Amplifluor HBV detection system is a cost-effective, high-throughput screening assay with sensitive target detection by amplification in standard thermocyclers followed by endpoint analysis using a fluorescence plate reader.

In contrast to the extensive precautions taken with current HBV assays, the Amplifluor system also minimizes false positives through the simultaneous amplification and detection of target DNA within a closed reaction vessel. More-sensitive and more cost-effective screening of blood pools can be achieved with the Amplifluor system by coupling PCR amplification with automated sample processing and automated endpoint detection.

The HBV Amplifluor assay will serve as a model for the development of future sensitive viral assays that are compatible with high-throughput screening of blood or blood products by endpoint analysis while retaining the option for quantitative real-time analysis.

References

1. KB Mullis and FA Faloona, "Specific Synthesis of DNA In Vitro via a Polymerase-Catalyzed Chain Reaction," Methods in Enzymology 155 (1987): 335–350.

2. J Compton, "Nucleic Acid Sequence-Based Amplification," Nature 350, no. 6313 (1991): 91–92.

3. GT Walker et al., "Strand Displacement Amplification: An Isothermal In Vitro DNA Amplification Technique," Nucleic Acids Research 20, no. 7 (1992): 1691–1696.

4. DC Thomas et al., "Amplification of Padlock Probes for DNA Diagnostics by Cascade Rolling Circle Amplification or the Polymerase Chain Reaction," Archives of Pathology and Laboratory Medicine 123, no. 12 (1999): 1170–1176.

5. T Horn et al., "Chemical Synthesis and Characterization of Branched Oligodeoxyribonucleotides (bDNA) for Use as Signal Amplifiers in Nucleic Acid Quantitation Assay," Nucleic Acids Research 25, no. 23 (1997): 4842–4849.

6. PM Holland et al., "Detection for Specific Polymerase Chain Reaction Product by Utilizing the 5' 3' Exonuclease Activity of Thermus aquaticus DNA Polymerase," Proceedings of the National Academy of Science (USA) 88, no. 16 (1991): 7276–7280.

7. GL Lee, CR Connell, and W Bloch, "Allelic Discrimination by Nick-Translation PCR with Fluorogenic Probes," Nucleic Acids Research 21, no. 16 (1993): 3761–3766.

8. S Tyagi and FR Kramer, "Molecular Beacons: Probes That Fluoresce upon Hybridization," Nature Biotechnology 14, no. 3 (1996): 303–308.

9. IA Nazarenko et al., "A Closed Tube Format for Amplification and Detection of DNA Based on Energy Transfer," Nucleic Acids Research 25, no. 12 (1997): 2516–2521.

10. IA Nazarenko et al., "Nucleic Acid Amplification Oligonucleotides with Molecular Energy Transfer Labels and Methods Based Thereon," U.S. Pats. 5,866,366; 6,090,592; 6,117,635; and 6,117,986.

11. N Gitlin, "Hepatitis B: Diagnosis, Prevention, and Treatment," Clinical Chemistry 43, no. 8 (1997): 1500–1506.

12. EL Murphy et al., "A Prospective Study of the Risk of Transfusion-Acquired Viral Infections," Transfusion Medicine 8, no. 3 (1998): 173–178.

13. SA Glynn et al., "Trends in Incidence and Prevalence of Major Transfusion-Transmissible Viral Infections in U.S. Blood Donors, 1991 to 1996," Journal of the American Medical Association 284, no. 2 (2000): 229–235.

14. BN Field et al., Virology, 3rd ed. (Philadelphia: Lippincott-Raven, 1996).

15. MM Manak, "Nucleic Acid Testing on Pooled Blood Donor Samples" (paper presented at the American Association of Blood Banks Annual Meeting, Denver, CO, October 18–22, 1997).

16. A Mangia et al., "Pathogenesis of Chronic Liver Disease in Patients with Chronic Hepatitis B Virus Infection without Serum HbeAg," Digestive Diseases and Sciences 41, no. 12 (1996): 2447–2452.

17. JM Jongerius et al., "New Hepatitis B Virus Mutant Form in a Blood Donor That Is Undetectable in Several Hepatitis B Surface Antigen Screening Assays," Transfusion 38, no. 1 (1998): 56–59.

18. S Lee et al., "Direct Detection of HBV Using the Gen-Probe Transcription-Mediated Amplification/Dual Kinetic Assay System" (poster presented at the American Association for Clinical Chemistry San Diego Conference: Nucleic Acid Technologies in Disease Detection, San Diego, November 17–19, 1999).

19. J Ji et al., "Simultaneous Genotyping HBV, HCV and HIV in Plasma Samples Using a Multiplex Microplate Capture Assay," Clinical Chemistry 45, no. 11 (1999): 2050.

20. H Uehara et al., "Detection of Telomerase Activity Utilizing Energy Transfer Primers: Comparison with Gel- and ELISA-Based Detection," Biotechniques 26, no. 3 (1999): 552–558.

Sandra K. Randall, PhD, is the director of research and development at Serologicals Corp. (Gaithersburg, MD); she can be reached via s.randall@intergenco.com. Jay Ji, PhD, is director of molecular biology; Bita Nakhai, PhD, is a product development project scientist; Nate Lawrence, PhD, is director of product development and technology transfer; and Mark Manak, PhD, is general manager at BBI Biotech Research Laboratories Inc. (Gaithersburg, MD); they can be reached via jji@bbii.com, bnakhai@bbii.com, nlawrence@bbii.com, and mmanak@bbii.com, respectively.

Copyright ©2002 IVD Technology