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Molecular Diagnostics and Genetics |
1 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT.
2 Department of Pathology, University of Utah Medical School, Salt Lake City, UT.
aAddress correspondence to this author at: Advanced Technology Group, ARUP, 500 Chipeta Way, Salt Lake City, UT 84108. Fax 801-584-5114; e-mail 18298{at}aruplab.com.
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Abstract |
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Methods: HCV RNA was purified and amplified in tubes coated with a solid-phase matrix that irreversibly bound nucleic acid during the extraction step. After reverse transcription-PCR, the amplicon was adsorbed to the original extraction matrix for purification and use in the subsequent sequencing reactions.
Results: The STETS method generated genotyping-quality sequence for a range of HCV titers from 500 to 6 000 000 IU/mL. If a viral sample was detected during real-time reverse transcription-PCR, it could be sequenced and genotyped. Read lengths >600 bases were observed with the STETS method. Mixed infections were detected and genotyped if at least 15% of the minor species was present. Combining the STETS method with consecutive sequencing provided a means of performing both forward and reverse sequencing in a single tube.
Conclusions: A single-tube nucleic acid extraction-to-sequencing method, which requires less time and labor than conventional methods, generates HCV sequence data that are equivalent to conventional methods and can be used to genotype HCV.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first step in any HCV genotyping protocol is to extract HCV genomic RNA from the virions present in the patient sample. Many nucleic acid extraction methods involve initial binding and then extraction from silica beads or filters. Commercially available methods for HCV RNA extraction commonly are either silica-based or use guanidine thiocyanate lysis followed by alcohol precipitation; all require centrifugation, dilution, or sample transfer (6)(11)(12)(13)(14).
In contrast, a nucleic acid extraction system that does not require centrifugation or precipitation and retains the isolated nucleic acid in the original extraction tube from lysis to amplicon generation has been developed (15)(16). The extraction tubes are coated with a solid-phase matrix that binds nucleic acids but not proteins. The binding of nucleic acid to the solid-phase matrix, which is based on hydrophilic and ionic interactions, depends on the buffer conditions. Guanidine buffers and NaOH will cause the nucleic acid to bind single-stranded to the matrix, whereas in phosphate buffers and water the majority of the nucleic acid is unbound and in the aqueous phase (16). The nucleic acid is irreversibly bound to the matrix during extraction, but remains accessible for amplification (15)(16). After PCR, the amplified products are present in solution and can be removed for sequencing, cloning, or other protocols (15). The extraction system has been used for Chlamydia trachomatis, Escherichia coli, and Cryptosporidium parvum detection (17)(18)(19), and nucleic acid can be extracted from many clinically relevant sources, such as blood and tissues (15). However, viral RNA extraction using this matrix has not been described in the literature.
During the development of the HCV RNA extraction protocol, we tested whether the solid-phase matrix could bind PCR products in addition to the already bound HCV RNA. The matrix was found to adsorb sufficient PCR amplicon to serve as a template for the sequencing reaction. Amplicon adsorption to the matrix provided a simple protocol for HCV lysis, HCV RNA purification, reverse transcription, PCR, amplicon purification, and generation of sequencing products within a single tube. This single-tube extraction-to-sequencing (STETS) method also allowed for a second sequencing reaction within the original extraction tube, with use of the opposite primer of the first reaction (referred to as consecutive sequencing), after the first set of sequencing reaction products were removed.
This report demonstrates that an immobilized, nucleic acid-binding matrix can isolate HCV RNA and generate PCR amplicon by reverse transcription-PCR (RT-PCR). In addition, the original extraction tube can be used for the forward and reverse sequencing reactions, eliminating all sample transfers from extraction to sequencing.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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conventional method for extraction to sequencing
HCV RNA extraction.
HCV RNA was extracted from plasma samples (140 µL) by use of QIAamp viral RNA Extraction Kit (Qiagen) according to manufacturer’s instructions. In brief, virions were lysed and the RNA genomes captured on the spin columns provided. The HCV RNA was washed and then eluted by incubating the spin column for 1 min in 60 µL of water followed by a 1-min centrifugation at 6000g at room temperature. The HCV RNA was stored at –20 °C.
Reverse transcription-PCR.
The 5'-UTR of the HCV RNA genome was amplified by use of the OneStep RT-PCR Kit (Qiagen). A sample volume of 2 µL of purified HCV RNA was added to a final RT-PCR reaction volume of 50 µL. The RT-PCR reaction contained 1x OneStep enzyme mixture and buffer with 0.32 mM each of dATP, dCTP, and dGTP; 0.64 mM dUTP (GeneAmp; Applied Biosystems) (20); 3.5 mM MgCl2; 0.25 µM each primer [forward, ME81 (5'-CTGCGGAACCGGTGAGTACACC-3'); reverse, KY78 (5'-CTCGCAAGCACCCTATCAGGCAGT-3'] (21); 0.25x SYBR Green I (Molecular Probes); 10 nM fluorescein (Bio-Rad); 0.01 U/µL uracil-DNA glycosylase (Roche); and 0.3 units/µL RNase inhibitor (Roche). The primers bound to conserved areas of the 5'-UTR to generate a 164-bp amplicon (from –31 to –194 of the HCV 5'-UTR; GenBank accession no. NC_004102). This amplicon contains variable regions of the 5'-UTR that can be used to determine HCV genotype (7)(10). RT-PCR was preformed on an iCycler iQTM real-time detection system (Bio-Rad) with the following conditions: the uracil-DNA glycosylase step was 50 °C for 10 min, the reverse transcription step was 55 °C for 30 min, and the PCR activation was 95 °C for 15 min. The 47 PCR cycles each consisted of three steps: denaturation at 95 °C for 30 s, annealing at 68 °C for 30 s, and extension at 72 °C for 60 s. Melting curves were generated after denaturation at 95 °C for 30 s and annealing at 65 °C for 30 s, followed by fluorescence acquisition every 10 s in 0.5 °C steps. Samples were then cooled to 65 °C for 30 s, then to 4 °C for storage.
Amplicon purification for sequencing.
PCR amplicons were purified by use of the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. Briefly, samples were bound to a purification column, washed, then eluted by centrifugation in 50 µL of elution buffer.
Conventional sequencing reaction preparation.
The sequencing reaction was prepared by mixing 8 µL of Big Dye Terminator Sequencing Mix, Version 1.1 (Applied Biosystems), 4 µL of primer at 0.8 pmol/µL (3.2 pmol), 4 µL of PCR-grade water, and 4 µL of purified PCR product.
stets method
HCV RNA extraction.
HCV RNA was purified from clinical plasma samples by use of extraction tubes coated with a nonsilica, nucleic acid-binding, solid-phase matrix (Xtra Amp® Extraction System-Series III; Xtrana, Inc.), according to manufacturer’s instructions as modified below (15). AVL lysis buffer (200 µL; Qiagen) containing 3 mmol/L dithiothreitol (Sigma) was added to the solid-phase matrix-coated extraction tubes (Xtra Amp tubes) 5 min before the plasma sample was added (50 µL). The samples were mixed by pipetting up and down 10 times with the pipette tip at the bottom of the tube. The samples were incubated at room temperature for 10 min with mixing (as above) at 5 min and at the end of incubation. Samples were then aspirated twice to discard all liquid. Tubes were washed twice with 200 µL of wash buffer (included in the reagent set from Xtrana) by mixing and aspirating as above. This wash procedure was repeated once with 200 µL of water. Tubes were stored dry at –20 °C or used immediately.
RT-PCR.
The 5'-UTR of the HCV RNA genome was amplified as stated in the conventional method, except that the purified HCV RNA was already bound to the matrix-coated extraction tubes. In addition, the primer concentrations were increased to 0.6 µM, and 5 µL of Amp Enhance Solution (included in the reagent set from Xtrana) was added.
A longer, 829-bp product (–194 to +635) was also amplified by use of the same forward primer as above and reverse primer 5'-AATACTCGAGTTAGGGCAATC-3', which binds in the core region [(22); GenBank M67463 and AF009606]. All reagent concentrations and reaction protocols were the same as for the 164-bp amplicon, except that 48 cycles were used, annealing was at 61 °C, and the extension time was 70 s.
At this stage the amplicon can be purified and sequenced as in the conventional method described above, or the amplicon can be adsorbed onto the solid-phase matrix (described below).
Amplicon purification by adsorption to the matrix.
After the PCR reaction, 50 µL of 1 mol/L NaOH was mixed (as described above) with 50 µL of the aqueous PCR amplicon within the original matrix-coated extraction tube. The mixture was incubated at room temperature for 10 min. After incubation, samples were again mixed and aspirated twice. Tubes were washed twice with 200 µL of the Wash Buffer (Xtrana) and once with 200 µL of water, and stored dry at 4 °C.
Sequencing reaction preparation.
The STETS method sequencing reaction contained 8 µL of Big Dye Terminator Sequencing Mix, Version 1.1 (Applied Biosystems), 4 µL of the sequencing primer (either forward primer ME81 or reverse primer KY78) at 8 pmol/µL (32 pmol), and 8 µL of PCR-grade water. All reagents were added to the bottom of the extraction tube containing the amplicon adsorbed to the matrix. Because vortex-mixing was found to adversely affect results, reagents were mixed as described previously.
Consecutive sequencing.
The sequencing reaction products were removed, and 50 µL of 0.5 mol/L NaOH was added and mixed in the empty extraction tubes that still contained amplicon bound to the matrix. After a 10-min incubation, the solution was mixed and discarded. The tubes were washed twice with wash buffer and once with water, and sequencing reagents were added as before.
cycle sequencing
Thermocycling conditions.
Sequencing reactions for both methods were processed in a GeneAmp 9700 thermocycler (Applied Biosystems). The GeneAmp 9700 ramp rate was set at 1 °C/s (9600 emulation mode). Sequencing products were generated over 25 cycles with the following conditions: denaturation at 96 °C for 10 s, primer annealing at 50 °C for 5 s, extension at 60 °C for 4 min, and a final 4 °C hold.
Sequencing reaction product purification.
Excess dye terminators were removed from the sequencing reaction products by use of Sephadex G-50 fine (Amersham Biosciences) in a multiscreen 96-well filtration plate (Millipore). After the addition of 300 µL of PCR-grade H2O to each well containing 45 µg of Sephadex G-50 fine, the plate was incubated at room temperature for 3 h. The interstitial water was removed by placing the filter plate over a standard microtiter tray and centrifuging at 900g for 2 min. Sequencing samples (20 µL) were loaded on the center of the column bed and then centrifuged again into a clean collection plate at 900g for 2 min. Samples were then dried in the DNA 120 microtiter plate Speed Vac (Savant Instruments Inc.).
Electrophoresis of sequencing reaction products.
Dried sequencing reaction products were resuspended in 20 µL of HiDi Formamide (Applied Biosystems), incubated at 95 °C for 2 min, and snap-cooled to 4 °C in a CoolSafeTM block (Diversified Biotech). Once cooled, the samples were allowed to warm to room temperature. Sequencing products were sequenced on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems) equipped with a 50-cm capillary array, using standard run conditions (2.5 h).
Data analysis.
Sequencing data were edited by use of Sequencher 4.1 for MAC. Editing consisted of aligning electronic sequencing data from primers ME81 (5') and KY78 (3') with the reference sequence, 5'-UTR HCV genotype 1a. Sequencing data from both directions were reviewed and edited to generate a consensus HCV sequence for the sample. The consensus sequence was assigned a HCV genotype by matching it to a sequence from a library of known HCV genotypes by use of MatchTools 1.0 software (Applied Biosystems).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The crossing thresholds for the solid-phase matrix amplifications were delayed two to three cycles later than conventional amplification.
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For both methods, the amplicons were purified with a Qiagen spin column and added to the sequencing reaction. Identical sequencing results were obtained in terms of signal strength, background fluorescence, quantity of ambiguous bases, and peak spacing (Fig. 2
, A and B). The 164-bp amplicon contained many genotype-specific nucleotides (7)(10). In a blinded study, 31 HCV clinical samples were correctly genotyped by use of the solid-phase matrix for extraction and amplification, including the common genotypes: 1a, 1b, 2b, and 3a.
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stets
Instead of removing the RT-PCR amplicon from the tube for sequencing (15), we added NaOH to the aqueous PCR product in the matrix-coated tube to irreversibly bind the amplicons, single-stranded, to the solid-phase matrix (amplicon adsorption). After washing, the sequencing reagents were added directly to an extraction tube with the matrix-adsorbed amplicon. Two tubes were usually used for sequence analysis: one for the forward primer and one for the reverse primer. Fig. 2
shows sequences generated from the STETS method (Fig. 2C
), the solid-phase matrix used for extraction and amplification only (Fig. 2B
), and the conventional method (Fig. 2A
). One discrepant call (A
C) was noted at nucleotide –136 in the STETS method (Fig. 2C
). This was caused by a nonspecific peak 
40 bases from the primer, which occasionally occurred with either primer. This peak always registered as a cytosine. The intensity of this nonspecific peak varied with the signal strength of the sequencing products and was very low or absent when the signal strength was high. HCV genotyping was not adversely affected by this nonspecific peak because it was located in an area not used for genotyping. Much less frequently, a second nonspecific cytosine peak was present in the sequence 75 bases from the primer. This second peak was of low intensity, and the sequence was read correctly through the interference.
When HCV clinical samples were serially diluted, extracted, and amplified by the STETS method, two of six samples at 1000 IU/mL did not generate amplicon, and only the samples with amplicon generated sequences. In a separate experiment, all six samples at 1000 IU/mL amplified, and only one of six samples at 500 IU/mL did not amplify (data not shown). When amplification occurred, the STETS method successfully produced sequence.
An 829-bp amplicon was used to test the quality and length of sequence obtained with the STETS method. A comparison of sequencing reactions performed either on the solid-phase matrix or by a conventional method is shown in Fig. 3
. Both methods generated sequences of equivalent quality to 600 bases, demonstrating the ability of the STETS method to generate and sequence larger amplicons.
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The matrix-coated extraction tubes can also be used for nucleic acid archiving (15)(16). Matrix-extracted HCV RNA could be stored dry at –20 °C for 1.5 months without affecting RT-PCR. Furthermore, amplicon bound to the tubes and stored dry at 4 °C for 2 months produced the same quality sequence as replicate samples used immediately (data not shown).
mixed infections
HCV mixed infections contain more than one genotype, and identifying minor HCV genotypes may change the duration of treatment (1)(3)(23). Two clinical HCV samples of genotypes 1a and 3a, each with a starting titer of 200 000 IU/mL, were combined in various percentages to determine the lowest amount of minor species detected by the STETS method. This method could detect minor species down to 15% of the population (data not shown). This sample would have a minor species HCV titer of 30 000 IU/mL in a background of the major species HCV titer of 170 000 IU/mL. A clinical mixed-infection HCV sample was sequenced by the STETS method and identified as a combination of 2b and 3a genotypes (Fig. 4
, top left panel). On the basis of the heights of the sequencing traces for the nucleotides that contained two peaks, the minor HCV species was present at 30–50% of the population.
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consecutive sequencing
The amplicon that was adsorbed to the solid-phase matrix after RT-PCR remained on the tube after the first sequencing reaction products were removed. The opposite primer was subsequently added along with new sequencing reagents for consecutive sequencing. The sequence background was slightly higher, but the sequence could still be used for HCV genotyping (Fig. 4
, consecutive sequencing in the bottom panels).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The STETS method amplified and sequenced all HCV samples tested with a titer >1000 IU/mL. Of samples with titers of 500-1000 IU/mL that were tested by the STETS method, 5 of 32 samples did not amplify. If the amplicon was detected during real-time RT-PCR, there was enough amplicon to generate sequence by the STETS method. HCV titer and genotype results (by methods stated in the Materials and Methods) from >6000 clinical samples submitted to ARUP were analyzed. Only 0.7% (44 of 6361) of these samples had a titer between 200 and 1000 IU/mL, and 20% could not be genotyped by conventional sequencing methods. Twelve percent (771 of 6361) of HCV clinical samples were <200 IU/mL, and the majority (97.4%) could not be genotyped. Less than 1% of HCV clinical samples that could be genotyped by conventional sequencing methods would not have been genotyped by the STETS method, using a conservative minimum titer of 1000 IU/mL for the STETS method. This limit of detection (1000 IU/mL) is similar to those reported in previous studies using other HCV 5'-UTR genotyping methods, Inno-LiPA and TRUGENE (24).
Mixed infections can be identified and genotyped by the STETS method. This method could detect a minor species down to 15% of the population or a minor species HCV titer of 30 000 IU/mL in a background of the major species HCV titer of 170 000 IU/mL. The minor species titer is well above our limiting titer for sequencing using the STETS method, but is not detected. Presumably, the minor species is overwhelmed by the quantity of RT-PCR and sequencing products from the major species; therefore, the nucleotide differences that indicate a minor species do not register above background. Our sensitivity to detect minor species approaches those published for HCV genotyping by TRUGENE sequencing (10%, or 10 000 IU/mL in 90 000 IU/mL), Inno-LiPA (5%, or 5000 IU/mL in 95 000 IU/mL), and conventional sequencing (>10–20%) (24)(25).
After the first set of sequencing products are removed, the matrix-coated extraction tube still contains the original PCR amplicons bound to the matrix. This allows for a second sequencing reaction (consecutive sequencing) performed with the opposite primer. To minimize background noise in the second sequencing reaction, we added NaOH to tether any leftover sequencing products or primers from the first reaction to the matrix. Consecutive sequencing produced both forward and reverse sequences from a single, matrix-coated extraction tube.
Previous studies have sought to eliminate amplicon purification between PCR and sequencing to reduce reagent cost and protocol time. Miller et al. (26) described a method based on different ratios of primers. After PCR, reaction products were added to the sequencing reaction without purification or additional primers. Another group used a similar method with a multicapillary assembly and small reaction volumes to streamline PCR and sequencing (27). This was accomplished with different ratios of primers for PCR and minimal volume transfer of PCR product (without purification) to the sequencing reaction. The first 70 bp could not be read because of overlap with the residual dye-terminators. Finally, direct exponential amplification and sequencing (DEXAS) uses sequential activation of PCR and sequencing polymerases to combine PCR and sequencing reactions (28)(29). DEXAS also uses dye-primer chemistry, requiring multiple sequencing reactions per sample. The STETS method has advantages over these methods because it includes all steps from nucleic acid purification to sequencing products within one tube and can use RNA as the template. The STETS method also contains uracil-DNA glycosylase to reduce possible contamination by previously made amplicon. The solid-phase matrix does not bind protein; therefore, the uracil-DNA glycosylase is eliminated at the amplicon adsorption step and cannot inhibit the sequencing reaction (20).
During the STETS method, the nucleic acid (RNA or DNA) is irreversibly bound to a solid-phase matrix, enabling all enzymes and buffers to be exchanged. Protocols can therefore be individually optimized for the RT-PCR and sequencing reactions. The quality of sequence obtained with the STETS method is similar to that obtained with conventional sequencing, and sequences close to the primer can be read (5–10 bases from the primer). The matrix-coated tubes can also be used for nucleic acid archiving because of the irreversible binding of nucleic acids to the solid-phase matrix (15)(16).
The STETS method does not require sample transfer at any step until sequencing product purification. This reduces processing time and the chance of sample labeling errors. The STETS method can easily be automated because only additions, mixing, and aspirations are required, with no centrifugation steps. In addition, use of the STETS method along with consecutive sequencing requires only one extraction tube for sequencing in both the forward and reverse directions.
In conclusion, the STETS method incorporates all steps from sample preparation to sequencing of reaction products in a single tube. This rapid protocol can generate sequence of the same quality as other, more time-consuming methods.
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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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-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med 1998;339:1485-1492.
2b plus ribavirin for 48 weeks or for 24 weeks versus interferon 2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International Hepatitis Interventional Therapy Group (IHIT). Lancet 1998;352:1426-1432.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
-2b with and without ribavirin for chronic hepatitis C. The Swedish Study Group. Lancet 1998;351:83-87.
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indicates an error in sequence attributable to a nonspecific peak sometimes present when the STETS method is used. Sequencing results for the reverse direction were of the same quality.
