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Microchip and capillary electrophoresis for quantitative analysis of hepatitis C virus based on RT-competitive PCR
Talanta 56 (2002) 323– 330 www.elsevier.com/locate/talanta
Microchip and capillary electrophoresis for quantitative analysis of hepatitis C virus based on RT-competitive PCR Kung-Chia Young b, Hsiang-Mei Lien a, Chun-Che Lin a, Ting-Tsung Chang c, Gwo-Bin Lee d, Shu-Hui Chen a,* b
a Department of Chemistry, National Cheng Kung Uni6ersity, Tainan, Taiwan, ROC Department of Medical Technology, National Cheng Kung Uni6ersity Hospital, Tainan, Taiwan, ROC c Department of Internal Medicine, National Cheng Kung Uni6ersity Hospital, Tainan, Taiwan, ROC d Department of Engineering Science, National Cheng Kung Uni6ersity, Tainan, Taiwan, ROC
Received 4 April 2001; received in revised form 28 May 2001; accepted 29 May 2001
Abstract A method to quantitatively perform reverse transcription-competitive PCR (RT-cPCR) of hepatitis C virus followed by both microchip and capillary electrophoretic separation and detection was described. In this method, HCV wild-type (WT) RNA extracted from serum was coretrotranscribed and coamplified with a constant amount of recombinant internal standard (IS) RNA which had the same primer binding region as the target RNA and was constructed by removing a centrally located 25-bp segment from the target template. A linear calibration curve was constructed by adding IS RNA at a constant concentration of 8000 copies ml − 1 into a series of RNA target standards ranging from 400 to 106 copies ml − 1. The amplified IS and target DNA were detected by both capillary and microchip electrophoresis via laser-induced fluorescence (LIF) using Cy5-labelled primer as the fluorescence probe. The method was further demonstrated for the quantitation of clinical patients with low, medium, and high viral titer and the results were found to be comparable to those determined by the commercial bDNA assay. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Microchip electrophoresis; Hepatitis C virus (HCV); Competitive PCR; Quantitation
1. Introduction Conventionally, slab gel electrophoresis is used for the analysis of PCR products including sizing, mutation, or polymorphism, but the technique is time-consuming, labor-intensive, and semi-quantitative. Due to the superior separation efficiency * Corresponding author. Fax: +886-6-274-0552. E-mail address: [email protected] (S.-H. Chen).
and speed in a miniaturized format, capillary electrophoresis (CE) is rapidly becoming an alternative tool for PCR analysis. As the electrophoresis technique advances to microchip devices, the use of microchip electrophoresis for DNA analysis is emerging as a promising method [1–3]. Moreover, the use of plastic material for the fabrication of microchips [4–6] is of great interest for clinical applications because the potentially low manufacturing costs may allow the microchips to be dis-
0039-9140/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 1 ) 0 0 5 9 8 - 7
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posable. In a previous study, we have demonstrated the use of CE for qualitative analysis of PCR product of hepatitis C virus (HCV) [7] and fragile X (CGG)n alleles [8] and the assay was found to be directly transferable to plastic microchip electrophoresis on PMMA substrate without tedious wall derivatization. In this study, a quantitative assay of HCV via competitive RTPCR was developed based on the same platform for electrophoretic separation and detection. Since circulating virus titer was recognized as an important parameter for monitoring the level of viral replication and predicting the outcome of antiviral treatment, viral genome quantitative assays have become increasingly desirable in clinical laboratories [9]. The well-known procedures for quantitation of HCV RNA include in-house competitive polymerase chain reaction (cPCR) [10– 25], real-time PCR [26– 28] and relatively more standardized Amplicor Monitor (Roche Diagnostics Systems, Basel, Switzerland) [29,30], branched-chain DNA (bDNA) (Bayer Diagnostics, Emeryville, USA) [31,32] and nucleic acid sequence-based amplification (Organon Teknika, Boxtel, The Netherlands) [33] assays. Most of these methods are based on hybridization detection in which the internal standard (IS) has the same length but a slightly different sequence compared to the target template. For electrophoretic detection, the detection of target and IS is based on length difference and hence the IS normally has a slightly different number of basepairs but the binding site for primers is unaffected. Design and characterization of RT-cPCR for HCV using capillary and microchip electrophoresis via laserinduced fluorescence (LIF) detection will be described in this work and the advantages and disadvantages of the method will be discussed as well.
2. Experimental section
2.1. Chemicals Tris[hydroxymethyl]aminomethane, (Tris) was purchased from Fluka (Buchs, Switzerland). Ethylenediaminetetraacetic acid, (EDTA) was ob-
tained from SIGMA (St. Louis, MO, USA). Boric acid was purchased from Janssen Chimica (Geel, Belgium). Hydroxypropyl methyl cellulose (HPMC) with a viscosity range of 80–120 cps (20 g l − 1 in H2O) was purchased from ALDRICH (St. Louis, MO, USA). All reagents were of the highest grade available. The Cy5-labelled PCR primers were synthesized by Sigma-Genosys Ltd. (The Woodlands, TX, USA). CE water was deionized distilled water filtered through a E-pure system which was purchased from Barnstead/ Thermolyne Corp. (Dubuque, IA, USA). The resistance of the water was more than 18.0 MV cm − 3.
2.2. Construction of target and IS template A 242-bp PCR product, obtained from a type1b HCV infected serum sample by RT-nested PCR was used as a target template. A set of nested primers from 5% noncoding region was used, including an outer primer pair of 400-base span: sense primer a (5%-TTGGGGGCGACACTCCACCATAG-3%, − 329– − 307 nt) and antisense primer b (5%-AACTTAACGTCCTGTGGGCGGCG-3%, 71 –49 nt) and an inner primer pair of 242-base span: sense primer c (5%-ACTCCACCATAGATCACTCC-3%, −318– − 299 nt) and antisense d (5%-AACACTACTCGGCTAGCAGT-3%, − 77– − 96 nt). A recombinant IS template was constructed by removing a 25-bp segment of the target template (spanning the region − 209 to − 185 nt). The deletion of the sequence was carried out with sense primer e (5%-CTGAAGCTTCGGTGAGTACACCGGAATTGCCAGG-3%, − 196– − 160 nt) and antisense primer f (5%-TCCCGGAAGCTTGGGTCCTGGAGGCTGCACGAC-3%, − 208– −240 nt). The underlined segments represent the Hind III recognition sequences created in the two primers. To generate the IS template, two separate PCR reactions were set up to create two short products from either end of the target template, with the primers c and f and the primers e and d, respectively. After being trimmed by Hind III, the digested PCR-A and PCR-B were ligated into a 217-bp DNA fragment. The final target and IS fragments were cloned into Srf I-cut
K.-C. Young et al. / Talanta 56 (2002) 323–330
pPCR-Script Amp SK( +) vector (Stratagene, La Jolla, CA, USA). The nucleotide sequences were confirmed with a cycle-sequencing protocol (DNA Terminator Cycle Sequencing Kit; Perkin– Elmer, USA) and analysis on an automatic sequencer (373A; Applied Biosystems, CA, USA).
2.3. RNA preparation For the preparation of synthetic target and IS RNA, the target and IS fragments were transcribed in vitro from linearized plasmid, using the RiboProbe® in vitro transcription kit (Promega). The absence of residual plasmid DNA was confirmed by DNase digestion (DNase I, 5 U mg − 1 DNA, 30 min, 37 °C) followed by PCR without reverse transcriptase used for cDNA synthesis and the obtained product was analyzed by agarose gel electrophoresis to ensure that only cRNA band was present. After enzyme digestion, the resulting cRNAs were then quantified from the measurement of absorption optical density (OD) at 260 nm. For the preparation of clinical samples, RNA was extracted from 100 ml of serum using the PUREscript™ RNA Isolation Kit (Gentra Systems Inc., Minneapolis, MN). The extracted RNA was dried and then redissolved in 12 ml of distilled water. The resulting solution was proceeded for RT-PCR through the same procedure as that for target RNA standards. Distilled water and normal sera were used as negative controls in each experiment.
2.4. Re6erse transcription and PCR amplification An aliquot 5 ml of IS at a constant concentration was mixed with 5 ml of each target RNA standards or each serum RNA samples prior to the RT reaction. The RNAs were then reverse transcribed into cDNA at 42 °C for 1 h, using 2.5 mM of antisense primer d, 500 mM of each dNTP, 20 unit (U) of human placental RNAse inhibitor (Promega) and 200 U moloney murine leukaemia virus reverse transcriptase (Promega) in a final volume of 40 ml containing 50 mM Tris– HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 and 10 mM dithiothreitol. Samples were then incubated at
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95 °C for 10 min for heat-inactivation of the enzymes. The first amplification of cDNA was performed in a reaction mixture of 50 ml containing 1/10 of the cDNA solution, 1×PCR-buffer (Amersham), 2.5 mM MgCl2, 200 mM of each dNTP, 20 pmoles of each primer c and d and 2.5 U Taqpolymerase (Amersham). Ten microliters of the first PCR-product were then reamplified with a primer pair of 145-base span: sense primer g (5%-TTCACGCAGAAAGCGTCTAG-3%, −279– 260 nt) and antisense primer h (5%-GTTGATCCAAGAAAGGACCC-3%, −135–154 nt) with a Cy5 coupling at the 5% end. Temperature cycling for both PCR runs comprised 35 cycles of DNA denaturation for 1 min at 94 °C, primer annealing for 1 min at 55 °C and elongation for 2 min at 72 °C. The PCR mixtures were then kept at 4 °C until analysis by both CE and microchip electrophoresis.
2.5. Con6entional CE instrumentation The experiments were performed using a Beckman P/ACE System 5500 (Beckman Instruments, Palo Alto, CA, USA) equipped with a home-built He –Ne laser (30 mW, LHR-991, Melles Griot, Carlsbad, CA, USA). The inner wall of fused-silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) with an inner diameter of 50 mm, outer diameter of 375 mm, effective length of 30 cm and total length of 37 cm was covalently bound with a non-cross-linked polyacrylamide according to the procedures described elsewhere [34]. Unless specified, samples were injected by a stream of nitrogen gas at 0.5 psi for 20 and 10 s for DNA marker and HCV amplicon, respectively. Separations were carried out in the reversed polarity mode (− 12 kV at the injector end which is equivalent to − 324 V cm − 1). Detection wavelength for LIF was set at 670 nm throughout the experiment, and the separation medium was composed of 1.5% HPMC in 100 mM TBE buffer (pH 8.2).
2.6. Microchip system A schematic diagram of the device configuration and dimensions is shown in Fig. 1. The
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channels were fabricated on PMMA plexiglass pieces approximately 1.5 cm in width× 4 cm in length and 1.5 mm in thickness using either a wire-imprinting [35] or heat-embossing method [36] as described and characterized previously [35,36]. As described in our previous study [7], the neutral hydrophilic nature of PMMA requires no surface modification for electrophoresis analysis of DNA molecules in the reversed polarity mode. For fused silica tubing, surface coating is normally required to minimize the adsorption of DNA molecules on the negative deprotonated silanol groups which provide electroosmotic flow (EOF) for the fluid delivery and separation when positive polarity mode is used. One power supplier (CZE 1000R, Spellman, Hauppauge, NY, USA) was utilized to furnish the loading and separation voltages and the power switching was controlled by a program written in LABVIEW (National Instruments, Austin, TX, USA) running on a PentiumIII 500 MHz computer. A sample volume of 2 –3 ml was pipetted to the chip reservoir and the sample loading was performed by applying − 300 V (−375 V cm − 1) to the injection
channel (between reservoir III and IV) for 0.2 min while keeping the separation channel (between reservoir I and II) floating. For the separation, − 1.2 kV (− 387 V cm − 1) was applied to the separation channel while keeping the buffer channel floating. The separation medium was composed of 1.5% HPMC in 100 mM TBE buffer (pH 8.2). Signals were detected on-microchip via LIF. The detection system was constructed through modifications of a commercial reflection microscope (Model BX40, Olympus, Tokyo, Japan). Briefly, a He–Ne laser beam with a wavelength of 632.8 nm (10 mW, LHR-991, Melles Griot, Carlsbad, CA, USA) was focused at a position 2 cm downstream from the cross section within the channel using a 50× (NA = 0.5) long-workingdistance objective. Fluorescence was collected by the objective and passed through a dichroic cube with band-pass filter, followed by spatial filtering prior to detection with a photomultiplier operated at − 650 V (R928, Hamamatsu, Tokyo, Japan). Amplified photoelectron pulses were converted to an analog signal and acquired by a commercial interface (Model 9524, SISC, Taipei, Taiwan) running on the same computer as used for voltage switching.
3. Results and discussion
Fig. 1. Channel configuration of the microchip. The buffer, analyte, and two waste reservoirs are indicated as I, III, II and IV, respectively.
Due to the separation power, the proposed detection platform using capillary or microchip electrophoresis via LIF allows the determination of PCR products from both target DNA and DNA IS in the same reaction vessel. As shown in Fig. 2, the IS and target DNA from the same reaction can be separated using HPMC as the sieving buffer. By comparing the migration time with those of a DNA digest marker, the bands corresponding to 120 and 145 bp were identified to be IS and target DNA, respectively. The early eluted peaks were due to the primer-dimers. Whereas, the later eluted peaks were not identified and could be due to the mis-amplified products. Apparently, the analysis time was greatly reduced from 7.5 min via CE (2A) to 50 s via microchip electrophoresis (2B) for a nearly equivalent ap-
K.-C. Young et al. / Talanta 56 (2002) 323–330
Fig. 2. Electropherogram of the amplified IS and target molecules by capillary (A) and microchip (B) device.
plied field, primarily due to a shorter separation length of the microchip. Moreover, it appears that even for the reduced separation length and separation time used for the microchip system, the resolution is competitive with conventional CE and adequate for this application. The quantitative PCR assay was carried out by coretrotranscribing and coamplifying samples containing target RNA concentrations varying from 437 to 106 copies ml − 1 with a constant amount of RNA IS in the concentration range of 8000–20 000 copies ml − 1. Following amplification, the products were determined by capillary or microchip electrophoresis. The detected signals which reflect the concentrations of amplification products from both target (Itarget) and IS (IIS) were plotted as a function of the logarithm of the concentration of target RNA molecules in the sample prior to amplification. In Fig. 3, it shows that the amplified DNA target remains constant for a small number of its initial molecules. However, as the target RNA increases and the PCR
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enters its plateau phase, a competitive binding for the primers occurs and the total amount of products including both IS and target molecules remain practically constant regardless of the starting amount of target RNA. As a consequence, an increase of target RNA within this concentration range results in suppression of the amplification of IS. Although the competitative reaction was designed to start from RNA level, it was found that the PCR results made no differences for IS added before or after RT reaction, indicating that both target and IS were independently retrotranscribed. The standard curves were constructed by plotting the logarithm of the intensity ratio of target and IS bands against the initial concentration of the target RNA molecules in the sample prior to RT-PCR. For each calibration curve, a blank which contained IS but no target RNA was prepared, amplified, and measured by the same protocol. As shown in Fig. 4, a good linear calibration curve ranging from 400 to 106 copies ml − 1 was obtained from either CE (4A) or microchip electrophoresis (4B) at a constant IS RNA concentration of 8000 copies ml − 1 prior to the reaction. The signal to noise ratio observed at 437 copies ml − 1 of the original target RNA concentration was around 3 for both CE and microchip electrophoresis and this sensitivity is comparable to that of bDNA assay. The relative standard deviation of the quantitation from three replicates of the standard was estimated to be less
Fig. 3. The attenuation of the detected fluorescence intensity of both IS and target molecules as a function of the intial target RNA concentration before the amplification. The initial IS concentration was 8000 copies ml − 1.
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Fig. 5. Microchip electropherograms of the amplified clinical serum samples with negative, low, and high virus concentrations.
Fig. 4. The calibration plots constructed from capillary electrophoresis (A) and microchip electrophoresis (B).
than 15% using the current assay. However, the standard deviation of between-day measurements was not investigated. Since the injection volume for CE or microchip electrophoresis is generally believed to be on the order of a few nanoliters of the amplified product, the detection limit in terms of copy number should be less than one copy from the pre-amplified serum. However, this sensitivity was gained largely owing to the nested PCR reaction that had concentrated the RNA in the original serum sample by several orders of magnitudes. Capillary or microchip electrophoresis is inherently limited by the tolerable injection volume and preconcentration steps such as sample stacking would help to increase the detection sensitivity. To assess the ability of the competitive RTPCR assay coupled with microchip electrophore-
sis/LIF detection for clinical quantitation of HCV, eight positive and one negative serum samples from clinical patients were processed together with a series of RNA standards by the current assay. These clinical samples had low to high viral titer as quantified by the commercial bDNA assay. As shown in Fig. 5, the intensity of the target band increased accordingly with the viral titer of the serum. There was no significant difference for the concentration determined by CE or microchip measurements. The virus concentration in the redissolved serum solution was calculated from the linear regression line of the calibration curve. The virus quantity in 1 ml of the original serum sample was then obtained by multiplying the calculated concentration in the re-dissolved solution with the volume conversion factor by assumming
Table 1 Comparisons of bDNA and cPCR-CE assays Patient number
bDNA (copies ml−1)
cPCR-CE (copies ml−1)
1 2 3 4 5 6 7 8 Negative
106.26 B105.30 B105.30 107.77 B105.30 106.66 107.20 107.01 0
106.18 105.00 105.17 108.11 105.12 106.42 106.88 107.80 0
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a 100% extraction yield. As shown in Table 1, it was found that the determined viral concentrations using the current assay were comparable to those using the bDNA assay. Although the analysis time for CE or microchip electrophoresis is within minutes including buffer equilibrium and washing, the cRT-PCR would take hours to finish, which has made this fast assay almost comparable to bDNA assay in terms of required time. However, the cost for cPCR-CE assay was estimated to be a hundred times less than that for bDNA assay.
4. Conclusion
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[7] [8] [9] [10] [11]
This work demonstrates a method to quantitatively perform reverse transcription-competitive PCR (RT-cPCR) of HCV followed by microchip and capillary electrophoretic separation and detection. Compared to the hybridization-based detection method, the electrophoretic separationbased assay demands less labor for washing and binding. However, the sensitivity is one of the most important concerns for clinical diagnostics. A preconcentration step such as sample stacking which is commonly applied in CE or the development of a more sensitive detection system will be important for a miniaturized system. Moreover, the greatest advantage of the electrophoretic chip platform is its potential for directly integrating sample processing steps such as PCR and extraction. These functions are currently explored in many laboratories [37,38] and some automatic instrumentation has become available recently. Hopefully, an automatic instrument together with a variety of user-friendly kits which do not require tedious method development will be widely accepted for clinical analysis in the near future.
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Acknowledgements Financial support from National Science Council of the Republic of China in Taiwan is greatly acknowledged.
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[28] M. Martell, J. Gomez, J.I. Esteban, S. Sauleda, J. Quer, B. Cabot, J. Clin. Microbiol. 37 (1999) 327. [29] E.P. Miskovsky, A.V. Carrella, K. Gutekunst, C.A. Sun, T.C. Quinn, D.L. Thomas, J. Clin. Microbiol. 34 (1996) 1975. [30] G. Gerken, T. Rothaar, M.G. Rumi, R. Soffredini, M. Trippler, M.J. Blunk, J. Clin. Microbiol. 38 (2000) 2210. [31] H.J. Alter, R. Sanchez-Pescador, M.S. Urdea, J.C. Wilber, R.J. Lagier, A.M. Di Bisceglie, J. Viral. Hepat. 2 (1995) 121. [32] J.M. Pawlotsky, M. Martinot-Peignoux, J.D. Poveda, A.
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Microchip and capillary electrophoresis for quantitative analysis of hepatitis C virus based on RT-competitive PCR Kung-Chia Young b, Hsiang-Mei Lien a, Chun-Che Lin a, Ting-Tsung Chang c, Gwo-Bin Lee d, Shu-Hui Chen a,* b
a Department of Chemistry, National Cheng Kung Uni6ersity, Tainan, Taiwan, ROC Department of Medical Technology, National Cheng Kung Uni6ersity Hospital, Tainan, Taiwan, ROC c Department of Internal Medicine, National Cheng Kung Uni6ersity Hospital, Tainan, Taiwan, ROC d Department of Engineering Science, National Cheng Kung Uni6ersity, Tainan, Taiwan, ROC
Received 4 April 2001; received in revised form 28 May 2001; accepted 29 May 2001
Abstract A method to quantitatively perform reverse transcription-competitive PCR (RT-cPCR) of hepatitis C virus followed by both microchip and capillary electrophoretic separation and detection was described. In this method, HCV wild-type (WT) RNA extracted from serum was coretrotranscribed and coamplified with a constant amount of recombinant internal standard (IS) RNA which had the same primer binding region as the target RNA and was constructed by removing a centrally located 25-bp segment from the target template. A linear calibration curve was constructed by adding IS RNA at a constant concentration of 8000 copies ml − 1 into a series of RNA target standards ranging from 400 to 106 copies ml − 1. The amplified IS and target DNA were detected by both capillary and microchip electrophoresis via laser-induced fluorescence (LIF) using Cy5-labelled primer as the fluorescence probe. The method was further demonstrated for the quantitation of clinical patients with low, medium, and high viral titer and the results were found to be comparable to those determined by the commercial bDNA assay. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Microchip electrophoresis; Hepatitis C virus (HCV); Competitive PCR; Quantitation
1. Introduction Conventionally, slab gel electrophoresis is used for the analysis of PCR products including sizing, mutation, or polymorphism, but the technique is time-consuming, labor-intensive, and semi-quantitative. Due to the superior separation efficiency * Corresponding author. Fax: +886-6-274-0552. E-mail address: [email protected] (S.-H. Chen).
and speed in a miniaturized format, capillary electrophoresis (CE) is rapidly becoming an alternative tool for PCR analysis. As the electrophoresis technique advances to microchip devices, the use of microchip electrophoresis for DNA analysis is emerging as a promising method [1–3]. Moreover, the use of plastic material for the fabrication of microchips [4–6] is of great interest for clinical applications because the potentially low manufacturing costs may allow the microchips to be dis-
0039-9140/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 1 ) 0 0 5 9 8 - 7
324
K.-C. Young et al. / Talanta 56 (2002) 323–330
posable. In a previous study, we have demonstrated the use of CE for qualitative analysis of PCR product of hepatitis C virus (HCV) [7] and fragile X (CGG)n alleles [8] and the assay was found to be directly transferable to plastic microchip electrophoresis on PMMA substrate without tedious wall derivatization. In this study, a quantitative assay of HCV via competitive RTPCR was developed based on the same platform for electrophoretic separation and detection. Since circulating virus titer was recognized as an important parameter for monitoring the level of viral replication and predicting the outcome of antiviral treatment, viral genome quantitative assays have become increasingly desirable in clinical laboratories [9]. The well-known procedures for quantitation of HCV RNA include in-house competitive polymerase chain reaction (cPCR) [10– 25], real-time PCR [26– 28] and relatively more standardized Amplicor Monitor (Roche Diagnostics Systems, Basel, Switzerland) [29,30], branched-chain DNA (bDNA) (Bayer Diagnostics, Emeryville, USA) [31,32] and nucleic acid sequence-based amplification (Organon Teknika, Boxtel, The Netherlands) [33] assays. Most of these methods are based on hybridization detection in which the internal standard (IS) has the same length but a slightly different sequence compared to the target template. For electrophoretic detection, the detection of target and IS is based on length difference and hence the IS normally has a slightly different number of basepairs but the binding site for primers is unaffected. Design and characterization of RT-cPCR for HCV using capillary and microchip electrophoresis via laserinduced fluorescence (LIF) detection will be described in this work and the advantages and disadvantages of the method will be discussed as well.
2. Experimental section
2.1. Chemicals Tris[hydroxymethyl]aminomethane, (Tris) was purchased from Fluka (Buchs, Switzerland). Ethylenediaminetetraacetic acid, (EDTA) was ob-
tained from SIGMA (St. Louis, MO, USA). Boric acid was purchased from Janssen Chimica (Geel, Belgium). Hydroxypropyl methyl cellulose (HPMC) with a viscosity range of 80–120 cps (20 g l − 1 in H2O) was purchased from ALDRICH (St. Louis, MO, USA). All reagents were of the highest grade available. The Cy5-labelled PCR primers were synthesized by Sigma-Genosys Ltd. (The Woodlands, TX, USA). CE water was deionized distilled water filtered through a E-pure system which was purchased from Barnstead/ Thermolyne Corp. (Dubuque, IA, USA). The resistance of the water was more than 18.0 MV cm − 3.
2.2. Construction of target and IS template A 242-bp PCR product, obtained from a type1b HCV infected serum sample by RT-nested PCR was used as a target template. A set of nested primers from 5% noncoding region was used, including an outer primer pair of 400-base span: sense primer a (5%-TTGGGGGCGACACTCCACCATAG-3%, − 329– − 307 nt) and antisense primer b (5%-AACTTAACGTCCTGTGGGCGGCG-3%, 71 –49 nt) and an inner primer pair of 242-base span: sense primer c (5%-ACTCCACCATAGATCACTCC-3%, −318– − 299 nt) and antisense d (5%-AACACTACTCGGCTAGCAGT-3%, − 77– − 96 nt). A recombinant IS template was constructed by removing a 25-bp segment of the target template (spanning the region − 209 to − 185 nt). The deletion of the sequence was carried out with sense primer e (5%-CTGAAGCTTCGGTGAGTACACCGGAATTGCCAGG-3%, − 196– − 160 nt) and antisense primer f (5%-TCCCGGAAGCTTGGGTCCTGGAGGCTGCACGAC-3%, − 208– −240 nt). The underlined segments represent the Hind III recognition sequences created in the two primers. To generate the IS template, two separate PCR reactions were set up to create two short products from either end of the target template, with the primers c and f and the primers e and d, respectively. After being trimmed by Hind III, the digested PCR-A and PCR-B were ligated into a 217-bp DNA fragment. The final target and IS fragments were cloned into Srf I-cut
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pPCR-Script Amp SK( +) vector (Stratagene, La Jolla, CA, USA). The nucleotide sequences were confirmed with a cycle-sequencing protocol (DNA Terminator Cycle Sequencing Kit; Perkin– Elmer, USA) and analysis on an automatic sequencer (373A; Applied Biosystems, CA, USA).
2.3. RNA preparation For the preparation of synthetic target and IS RNA, the target and IS fragments were transcribed in vitro from linearized plasmid, using the RiboProbe® in vitro transcription kit (Promega). The absence of residual plasmid DNA was confirmed by DNase digestion (DNase I, 5 U mg − 1 DNA, 30 min, 37 °C) followed by PCR without reverse transcriptase used for cDNA synthesis and the obtained product was analyzed by agarose gel electrophoresis to ensure that only cRNA band was present. After enzyme digestion, the resulting cRNAs were then quantified from the measurement of absorption optical density (OD) at 260 nm. For the preparation of clinical samples, RNA was extracted from 100 ml of serum using the PUREscript™ RNA Isolation Kit (Gentra Systems Inc., Minneapolis, MN). The extracted RNA was dried and then redissolved in 12 ml of distilled water. The resulting solution was proceeded for RT-PCR through the same procedure as that for target RNA standards. Distilled water and normal sera were used as negative controls in each experiment.
2.4. Re6erse transcription and PCR amplification An aliquot 5 ml of IS at a constant concentration was mixed with 5 ml of each target RNA standards or each serum RNA samples prior to the RT reaction. The RNAs were then reverse transcribed into cDNA at 42 °C for 1 h, using 2.5 mM of antisense primer d, 500 mM of each dNTP, 20 unit (U) of human placental RNAse inhibitor (Promega) and 200 U moloney murine leukaemia virus reverse transcriptase (Promega) in a final volume of 40 ml containing 50 mM Tris– HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 and 10 mM dithiothreitol. Samples were then incubated at
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95 °C for 10 min for heat-inactivation of the enzymes. The first amplification of cDNA was performed in a reaction mixture of 50 ml containing 1/10 of the cDNA solution, 1×PCR-buffer (Amersham), 2.5 mM MgCl2, 200 mM of each dNTP, 20 pmoles of each primer c and d and 2.5 U Taqpolymerase (Amersham). Ten microliters of the first PCR-product were then reamplified with a primer pair of 145-base span: sense primer g (5%-TTCACGCAGAAAGCGTCTAG-3%, −279– 260 nt) and antisense primer h (5%-GTTGATCCAAGAAAGGACCC-3%, −135–154 nt) with a Cy5 coupling at the 5% end. Temperature cycling for both PCR runs comprised 35 cycles of DNA denaturation for 1 min at 94 °C, primer annealing for 1 min at 55 °C and elongation for 2 min at 72 °C. The PCR mixtures were then kept at 4 °C until analysis by both CE and microchip electrophoresis.
2.5. Con6entional CE instrumentation The experiments were performed using a Beckman P/ACE System 5500 (Beckman Instruments, Palo Alto, CA, USA) equipped with a home-built He –Ne laser (30 mW, LHR-991, Melles Griot, Carlsbad, CA, USA). The inner wall of fused-silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) with an inner diameter of 50 mm, outer diameter of 375 mm, effective length of 30 cm and total length of 37 cm was covalently bound with a non-cross-linked polyacrylamide according to the procedures described elsewhere [34]. Unless specified, samples were injected by a stream of nitrogen gas at 0.5 psi for 20 and 10 s for DNA marker and HCV amplicon, respectively. Separations were carried out in the reversed polarity mode (− 12 kV at the injector end which is equivalent to − 324 V cm − 1). Detection wavelength for LIF was set at 670 nm throughout the experiment, and the separation medium was composed of 1.5% HPMC in 100 mM TBE buffer (pH 8.2).
2.6. Microchip system A schematic diagram of the device configuration and dimensions is shown in Fig. 1. The
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channels were fabricated on PMMA plexiglass pieces approximately 1.5 cm in width× 4 cm in length and 1.5 mm in thickness using either a wire-imprinting [35] or heat-embossing method [36] as described and characterized previously [35,36]. As described in our previous study [7], the neutral hydrophilic nature of PMMA requires no surface modification for electrophoresis analysis of DNA molecules in the reversed polarity mode. For fused silica tubing, surface coating is normally required to minimize the adsorption of DNA molecules on the negative deprotonated silanol groups which provide electroosmotic flow (EOF) for the fluid delivery and separation when positive polarity mode is used. One power supplier (CZE 1000R, Spellman, Hauppauge, NY, USA) was utilized to furnish the loading and separation voltages and the power switching was controlled by a program written in LABVIEW (National Instruments, Austin, TX, USA) running on a PentiumIII 500 MHz computer. A sample volume of 2 –3 ml was pipetted to the chip reservoir and the sample loading was performed by applying − 300 V (−375 V cm − 1) to the injection
channel (between reservoir III and IV) for 0.2 min while keeping the separation channel (between reservoir I and II) floating. For the separation, − 1.2 kV (− 387 V cm − 1) was applied to the separation channel while keeping the buffer channel floating. The separation medium was composed of 1.5% HPMC in 100 mM TBE buffer (pH 8.2). Signals were detected on-microchip via LIF. The detection system was constructed through modifications of a commercial reflection microscope (Model BX40, Olympus, Tokyo, Japan). Briefly, a He–Ne laser beam with a wavelength of 632.8 nm (10 mW, LHR-991, Melles Griot, Carlsbad, CA, USA) was focused at a position 2 cm downstream from the cross section within the channel using a 50× (NA = 0.5) long-workingdistance objective. Fluorescence was collected by the objective and passed through a dichroic cube with band-pass filter, followed by spatial filtering prior to detection with a photomultiplier operated at − 650 V (R928, Hamamatsu, Tokyo, Japan). Amplified photoelectron pulses were converted to an analog signal and acquired by a commercial interface (Model 9524, SISC, Taipei, Taiwan) running on the same computer as used for voltage switching.
3. Results and discussion
Fig. 1. Channel configuration of the microchip. The buffer, analyte, and two waste reservoirs are indicated as I, III, II and IV, respectively.
Due to the separation power, the proposed detection platform using capillary or microchip electrophoresis via LIF allows the determination of PCR products from both target DNA and DNA IS in the same reaction vessel. As shown in Fig. 2, the IS and target DNA from the same reaction can be separated using HPMC as the sieving buffer. By comparing the migration time with those of a DNA digest marker, the bands corresponding to 120 and 145 bp were identified to be IS and target DNA, respectively. The early eluted peaks were due to the primer-dimers. Whereas, the later eluted peaks were not identified and could be due to the mis-amplified products. Apparently, the analysis time was greatly reduced from 7.5 min via CE (2A) to 50 s via microchip electrophoresis (2B) for a nearly equivalent ap-
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Fig. 2. Electropherogram of the amplified IS and target molecules by capillary (A) and microchip (B) device.
plied field, primarily due to a shorter separation length of the microchip. Moreover, it appears that even for the reduced separation length and separation time used for the microchip system, the resolution is competitive with conventional CE and adequate for this application. The quantitative PCR assay was carried out by coretrotranscribing and coamplifying samples containing target RNA concentrations varying from 437 to 106 copies ml − 1 with a constant amount of RNA IS in the concentration range of 8000–20 000 copies ml − 1. Following amplification, the products were determined by capillary or microchip electrophoresis. The detected signals which reflect the concentrations of amplification products from both target (Itarget) and IS (IIS) were plotted as a function of the logarithm of the concentration of target RNA molecules in the sample prior to amplification. In Fig. 3, it shows that the amplified DNA target remains constant for a small number of its initial molecules. However, as the target RNA increases and the PCR
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enters its plateau phase, a competitive binding for the primers occurs and the total amount of products including both IS and target molecules remain practically constant regardless of the starting amount of target RNA. As a consequence, an increase of target RNA within this concentration range results in suppression of the amplification of IS. Although the competitative reaction was designed to start from RNA level, it was found that the PCR results made no differences for IS added before or after RT reaction, indicating that both target and IS were independently retrotranscribed. The standard curves were constructed by plotting the logarithm of the intensity ratio of target and IS bands against the initial concentration of the target RNA molecules in the sample prior to RT-PCR. For each calibration curve, a blank which contained IS but no target RNA was prepared, amplified, and measured by the same protocol. As shown in Fig. 4, a good linear calibration curve ranging from 400 to 106 copies ml − 1 was obtained from either CE (4A) or microchip electrophoresis (4B) at a constant IS RNA concentration of 8000 copies ml − 1 prior to the reaction. The signal to noise ratio observed at 437 copies ml − 1 of the original target RNA concentration was around 3 for both CE and microchip electrophoresis and this sensitivity is comparable to that of bDNA assay. The relative standard deviation of the quantitation from three replicates of the standard was estimated to be less
Fig. 3. The attenuation of the detected fluorescence intensity of both IS and target molecules as a function of the intial target RNA concentration before the amplification. The initial IS concentration was 8000 copies ml − 1.
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Fig. 5. Microchip electropherograms of the amplified clinical serum samples with negative, low, and high virus concentrations.
Fig. 4. The calibration plots constructed from capillary electrophoresis (A) and microchip electrophoresis (B).
than 15% using the current assay. However, the standard deviation of between-day measurements was not investigated. Since the injection volume for CE or microchip electrophoresis is generally believed to be on the order of a few nanoliters of the amplified product, the detection limit in terms of copy number should be less than one copy from the pre-amplified serum. However, this sensitivity was gained largely owing to the nested PCR reaction that had concentrated the RNA in the original serum sample by several orders of magnitudes. Capillary or microchip electrophoresis is inherently limited by the tolerable injection volume and preconcentration steps such as sample stacking would help to increase the detection sensitivity. To assess the ability of the competitive RTPCR assay coupled with microchip electrophore-
sis/LIF detection for clinical quantitation of HCV, eight positive and one negative serum samples from clinical patients were processed together with a series of RNA standards by the current assay. These clinical samples had low to high viral titer as quantified by the commercial bDNA assay. As shown in Fig. 5, the intensity of the target band increased accordingly with the viral titer of the serum. There was no significant difference for the concentration determined by CE or microchip measurements. The virus concentration in the redissolved serum solution was calculated from the linear regression line of the calibration curve. The virus quantity in 1 ml of the original serum sample was then obtained by multiplying the calculated concentration in the re-dissolved solution with the volume conversion factor by assumming
Table 1 Comparisons of bDNA and cPCR-CE assays Patient number
bDNA (copies ml−1)
cPCR-CE (copies ml−1)
1 2 3 4 5 6 7 8 Negative
106.26 B105.30 B105.30 107.77 B105.30 106.66 107.20 107.01 0
106.18 105.00 105.17 108.11 105.12 106.42 106.88 107.80 0
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a 100% extraction yield. As shown in Table 1, it was found that the determined viral concentrations using the current assay were comparable to those using the bDNA assay. Although the analysis time for CE or microchip electrophoresis is within minutes including buffer equilibrium and washing, the cRT-PCR would take hours to finish, which has made this fast assay almost comparable to bDNA assay in terms of required time. However, the cost for cPCR-CE assay was estimated to be a hundred times less than that for bDNA assay.
4. Conclusion
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This work demonstrates a method to quantitatively perform reverse transcription-competitive PCR (RT-cPCR) of HCV followed by microchip and capillary electrophoretic separation and detection. Compared to the hybridization-based detection method, the electrophoretic separationbased assay demands less labor for washing and binding. However, the sensitivity is one of the most important concerns for clinical diagnostics. A preconcentration step such as sample stacking which is commonly applied in CE or the development of a more sensitive detection system will be important for a miniaturized system. Moreover, the greatest advantage of the electrophoretic chip platform is its potential for directly integrating sample processing steps such as PCR and extraction. These functions are currently explored in many laboratories [37,38] and some automatic instrumentation has become available recently. Hopefully, an automatic instrument together with a variety of user-friendly kits which do not require tedious method development will be widely accepted for clinical analysis in the near future.
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Acknowledgements Financial support from National Science Council of the Republic of China in Taiwan is greatly acknowledged.
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