Accepted Manuscript Title: COMPARISON OF FOUR METHODS OF GENOTYPING IL28B POLYMORPHISMS IN CHRONIC HEPATITIS C PATIENTS Author: Nath´alia Delvaux Vanessa Duarte da Costa Maristella Matos da Costa Elisabeth Lampe PII: DOI: Reference:
S0166-0934(15)00130-5 http://dx.doi.org/doi:10.1016/j.jviromet.2015.04.001 VIRMET 12765
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
26-11-2014 1-4-2015 2-4-2015
Please cite this article as: http://dx.doi.org/10.1016/j.jviromet.2015.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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COMPARISON OF FOUR METHODS OF GENOTYPING IL28B POLYMORPHISMS
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IN CHRONIC HEPATITIS C PATIENTS
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Nathália Delvauxa, Vanessa Duarte da Costaa, Maristella Matos da Costaa, Elisabeth
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Lampea
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Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, RJ, Brazil
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*Corresponding author: Nathália Delvaux, Laboratório Nacional de Hepatites Virais,
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Instituto Oswaldo Cruz - FIOCRUZ. Avenida Brasil, 4365 - Manguinhos - Código
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Postal: 21040-900. Rio de Janeiro, RJ, Brazil. Telephone: (+55) 21 2562 - 1894. E-
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mails:
and
[email protected]
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SUMMARY
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Background: Single nucleotide polymorphisms (SNPs) of the interleukin 28B
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(IL28B) gene are associated with viral clearance and treatment response in hepatitis
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C virus (HCV) infection; however, most of the available SNP genotyping methods are
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expensive.
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Aims: This study sought to evaluate the cost effectiveness of four methods used to
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genotype the rs12979860 and rs8099917 SNPs of the IL28B gene.
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Methods: Tetra-primer amplification-refractory mutation system-polymerase chain
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reaction (ARMS-PCR), restriction fragment length polymorphism (RFLP), quantitative
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(q) PCR and direct sequencing methods were evaluated in terms of specificity, cost
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and run time in 281 blood samples obtained from chronic HCV patients.
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Results: In ARMS-PCR method, the primers designed to target both SNPs produced
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PCR fragments of specific sizes that distinguished the alleles of rs12979860 and
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rs8099917. In RFLP, the band profile allowed the distinction between genotypes. The
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qPCR was the faster and easier to perform. Validation by nucleotide sequencing
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showed 100% agreement among the three methods. The cost for a single reaction
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was lowest for ARMS-PCR, followed in turn by RFLP, qPCR and sequencing.
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Conclusions: The methodology described for the ARMS-PCR showed the most
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favorable cost-benefit ratio. Moreover, this approach is fast and simple, requiring only
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equipment that is commonly used in molecular diagnosis, which is an essential
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parameter for use in developing countries where laboratories have scarce financial
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resources.
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Key
words:
IL28B,
SNP,
HCV,
Brazil,
ARMS-PCR
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The treatment of chronic hepatitis C virus (HCV) infection, despite recent
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progress following the introduction of direct-acting antiviral (DAA) regimens, remains
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a great challenge in terms of cost effectiveness. Thus, the rapid identification of
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sustained virologic response (SVR) predictors remains a major target in HCV
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research. Two single nucleotide polymorphisms (SNPs) rs12979860 and rs8099917,
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in close proximity to the interleukin 28B (IL28B) gene, have reported associations
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with viral clearance and treatment response to HCV infection (Ge, et al., 2009;
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Tanaka et al., 2009). The IL28B gene encodes interleukin 28, a cytokine belonging to
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the IFN-λ family that is involved in the regulation of the immune response against
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viral infections (Dellgren et al., 2009; Li et al., 2009). The SVR rate with PEG-
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IFN/RBV therapy in HCV-1 infected patients is two-fold higher in individuals with the
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rs12979860 CC genotype than the CT or TT (70-80% vs. 30-40%) (Chen et al., 2011;
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Thompson et al., 2010). Similarly, the SVR is higher in individuals with the rs8099917
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TT genotype compared to the TT or GT+GG genotype (81% vs. 59%) (Sakamoto et
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al., 2011). Several clinical studies have validated these findings and, IL28B
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genotyping has become an important tool to assist in clinical decisions regarding the
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most appropriate therapeutic regimen. Furthermore, IL28B genotyping has shown
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that it may be useful as a first-generation DAA approach for identifying patients who
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can be treated successfully with a shorter and simpler treatment scheme (Jacobson
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et al., 2011; Poordad et al., 2011). Additionally, the impact of the IL28B CC genotype
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(rs12979860) was observed in HCV-1a infected patients undergoing IFN-free
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combination therapy (58 to 84% among patients with IL28B CC vs. 33 to 64% in
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patients with non-CC genotypes) (Chu et al., 2011; Zeuzem et al., 2012, 2013).
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IL28B genotyping can be determined using diverse methods, such as DNA
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sequencing, Taqman assays, PCR-RFLP, and DNA high-performance liquid
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Chromatography (DHPLC) (Fiorina et al., 2012; Medrano and Oliveira, 2014). These
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methods have different characteristics, including equipment needs, cost and
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technical knowledge. However, some of the methods are excessively expensive, and
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their high cost of implementation limits their use, especially in developing countries
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(Ferreira et al., 2013; Galmozzi et al., 2011; Medrano and Oliveira, 2014). Aiming to
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obtain a cheap and simple test to determine IL28B genotype, in house protocols for
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tetra-primer amplification-refractory mutation system-polymerase chain reaction
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(ARMS-PCR) and for restriction fragment length polymorphism (RFLP) were
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optimized in this study. Commercial qPCR method was also used and direct
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sequencing was used to validate these techniques. Additionally, the four methods
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used to determining the IL28B polymorphisms (rs12979860 and rs8099917) in
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patients with HCV infection were evaluated in terms of specificity, cost and run time.
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ARMS-PCR, RFLP, quantitative (q) PCR and direct sequencing methods were
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carried out in 281 blood samples (108 males, aged 56.0 ± 10.9 years) obtained from
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chronic HCV patients (positive anti-HCV antibody and detectable HCV RNA in serum
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samples of patients with infection for more than 6 months). The local Ethical
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Committee (CEP Nº 297.459) approved this study. Genomic DNA was extracted from
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200 µL of whole blood using the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany),
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according to the manufacturer’s directions, and stored at -20°C.
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In ARMS-PCR assay, to design the primers to target the two SNPs, we used the
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program
developed
by
Ye
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http://primer1.soton.ac.uk/primer1.html. The limiting of fragment sizes was chosen
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within the range of 100–300 bp with a ratio of allelic bands of 1.5. Default settings
et
al.
(2001),
available
at
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were used for the other parameters. For rs12979860, the forward primer (FIT 860)
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was designed to hybridize to the genomic sequence with the T allele, and the reverse
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primer (RIC 860) was designed to hybridize with sequences containing the C allele.
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The same procedure was conducted for rs8099917 (FIG 917 inner - G allele, RIT 917
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inner - T allele). The primers designed for ARMS-PCR assay are showed in table 1.
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PCRs for both SNPs were performed in a volume of 20 µL. Different amplification
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conditions (annealing temperatures, PCR cycle protocols and primer concentrations)
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were assessed to ensure proper formation of all the fragments. For rs12979860 the
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most appropriated PCR conditions were: 95°C for 15 min followed by 35 cycles of
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94°C for 30 s, 64°C for 30 s, 72°C for 1 min and 72°C for 10 min. For rs8099917, the
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best results were obtained with annealing temperature of 55°C using the same
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cycling conditions. The PCR products were separated by standard electrophoresis on
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2.5% agarose gels containing gel red dye (Biotium Inc., Hayward, CA), with a 100 bp
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molecular weight marker (Promega, Madison, Wisconsin, USA).
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The amplification results showed that the ARMS-PCR method distinguishes
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successfully between the two different SNPs. In the rs12979860 genotyping method,
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the outer primer pairs produced a band of 277 bp. The homozygote TT could be
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distinguished by an additional band of 198 bp, the homozygote CC could be
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distinguished by a band of 132 bp and heterozygote C and T alleles produced
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fragments of 132 bp and 198 bp, respectively. The genotypes of rs8099917 could be
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differentiated according to the following band profiles: TT - 437 bp and 295 bp; GG -
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437 bp and 197 bp; and TG - 437 bp, 295 bp and 197 bp. Figure 1A shows an
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agarose gel representing ARMS-PCR profiles for SNP rs12979860, and Figure 1B
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shows the results for SNP rs8099917.
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For RFLP, two pairs of primers were designed for each IL28B gene SNP (Table
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1). The primer pairs 860F/860R were designed to amplify a fragment of 242 bp for
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the rs12979860, and the primer pairs 917F/917R were designed to amplify a
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fragment of 539 bp for rs8099917. The PCRs were performed in separate tubes
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containing 0.2 pmol of the respective primer pairs. For rs12979860, the PCR
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conditions were as follows: 95°C for 15 min, followed by 35 cycles of 95°C for 1 min,
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58°C for 45 s, 72°C for 45 s and 72°C for 3 min. Amplified PCR products were
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digested with the restriction enzyme BstUI (10 U/rxn; New England Biolabs) at 37°C
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for 3.5 h. For rs8099917, PCR was conducted as follows: 95°C for 15 min; 10 cycles
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of 95°C for 1 min, 50°C for 45 s, and 72°C for 45 s; and 25 cycles of 94°C for 30 s,
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55°C for 30 s, 72°C for 30 s and 72°C for 3 min. The amplified PCR products were
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digested with the restriction enzyme Tsp45I (2 U/rxn; New England Biolabs) at 65°C
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for 3.5 h. The digested products of both SNPs were separated by standard
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electrophoresis on 3% agarose gels containing gel red dye (Biotium Inc., Hayward,
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CA), alongside 100 bp Molecular Weight (Promega, Madison, Wisconsin, USA). The
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242 bp PCR fragment obtained with the primers designed for typing rs12979860
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digested with BstUI produced the following band profiles: 3 fragments of 25 bp, 82 bp
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and 135 bp in individuals with the CC genotype; 2 fragments of 82 bp and 160 bp in
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individuals with the TT genotype; and 4 fragments of 25 bp, 82 bp, 135 bp and 160
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bp in individuals with the heterozygote CT genotype (Figure 1C). The enzymatic
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digestion of the 539 bp rs8099917 fragment with Tsp45I, produced a band profiles
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that allowed the distinction between the GG (39 bp, 214 bp and 286 bp), TG (39 bp,
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214 bp, 286 bp and 325 bp) and TT (214 bp and 325 bp) genotypes (Figure 1D).
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qPCR was performed using a commercial kit from Roche Life Technologies (TIB
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MOLBIOL GmbH, Berlin, Germany), according to the manufacturer’s instructions.
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The genotyping results for rs12979860 and rs8099917 with the qPCR method could
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distinguish between the respective SNP genotypes according the fluorescence
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profile. Direct sequencing after PCR amplification was done with the same primers as
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those used for the RFLP for each polymorphism. The PCR products were purified
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using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) and submitted to
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nucleotide sequencing reactions in both directions using the Big Dye Terminator kit
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3.1 (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s
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instructions, followed by analysis on the ABI 3730 DNA automated sequencer
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(Applied Biosystems, Foster City, CA, USA). In sequencing chromatogram,
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sequences that showed single peaks were considered as homozygous, whereas
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those that showed double overlapping peaks were interpreted as heterozygous. All
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results obtained agreed 100% with the other three methods used in this study. The
149
distribution of genotypes according to the methods used in this study is shown in
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Table 2.
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To estimate the cost of each technique, the price of reagents (enzymes, buffers,
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agarose, dyes, and other specific reagents for each procedure) and disposable
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materials (tips, polystyrene tubes, gloves) used in each of the methods was
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calculated based on currently available commercial prices. Other indirect costs were
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not included, such as equipment maintenance and human resources. Thus, the price
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of a single reaction for ARMS-PCR, RFLP, qPCR and direct sequencing was
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respectively: US$19.40; US$27.80; US$22.90 and US$202.80. The assay that
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requires more execution time was direct sequencing (2520 minutes or more),
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followed by RFLP (570 minutes), ARMS-PCR (300 minutes) and commercial qPCR
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(130 minutes).
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ARMS-PCR methodology showed the lowest cost and easiest execution, as well
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as the second fastest run time. In addition, this method presents a wide convenience
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of execution; it requires only equipment that is routinely used in most laboratories
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that perform molecular biology assessments, such as a thermal cycler and
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electrophoresis apparatus. In this technique, the high specificity of the reaction relies
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on the 3’ terminus mismatch and the position-2 (second to the terminal) mismatch
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from the 3’ terminus of the same allele-specific primer. This last mismatch
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destabilizes the base paring between the primers and their corresponding non-target
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templates and increases the specificity of the reaction by eliminating false-positive
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results (Ye et al., 2001). This method uses four primers in a single PCR reaction; two
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non-allele-specific primers (outer primers) amplify the region that comprises the SNP,
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and as the outer primer fragment is produced, it serves as a template for the two
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allele-specific primers (inner primers) that generate allele-specific fragments. By
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placing the outer primers at different distances from the SNP, the two allele-specific
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fragments can be distinguished by their different sizes in an agarose gel (Medrano
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and de Oliveira, 2014; Ye et al., 2001).
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The RFLP technique was the second most expensive method and required a
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longer run-time. This method also requires two stages: PCR reaction and incubation
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with restriction enzymes. Furthermore, it requires two electrophoresis runs: one for
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analysis of the PCR amplicons and the other for analyzing the band profiles after
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enzymatic digestion that takes more than 3 h. In addition, restriction enzymes are
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high in cost and are extremely unstable. On the other hand, despite its higher cost,
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the equipments required for RFLP are routinely used in most molecular biology
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laboratories (thermal cycler, electrophoresis apparatus and water bath) and the
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training for this technique and interpretation of the results is not complex. Thus, even
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with disadvantages in several issues, the RFLP technique is, along with ARMS-PCR,
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very accessible to laboratories with few financial resources. Among the four methods evaluated in this study, qPCR was the fastest and
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easiest to perform. Moreover, this method is sensitive and specific due to Taqman
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probe technology. qPCR was the second least expensive technique, even though it
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uses a commercial kit. However, this method requires a specific and expensive
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thermal cycler that is not available in many laboratories and requires additional
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training for the interpretation and analysis of data.
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Direct sequencing is considered the gold standard and was used in this study to
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validate the results obtained with the other techniques. This method is the most time-
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consuming and laborious of the procedures evaluated. The number of steps required
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is large, including two PCR rounds, agarose gel electrophoresis, purification of PCR
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products, and diverse handling steps, until the sequence is obtained as a
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chromatogram. Also, it is necessary to have good knowledge of sequencing
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programs to analyze the data, especially in the heterozygous case. Moreover, the
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equipment is extremely expensive, and the maintenance cost is high because it
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demands several specific reagents. In the present work, the results of all three
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methods were 100% concordant with the results of direct sequencing.
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In conclusion, all methods tested were specific for genotyping SNPs rs12979860
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and rs8099917 of the IL28B gene. However, ARMS-PCR showed the best results
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according to the cost-benefit analysis. This approach represents a simple, fast and
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cost-effective method that involves a single PCR reaction followed by gel
208
electrophoresis. Therefore, this technique is easy to use in a routine molecular
209
diagnostic setting, with minimum equipment requirements. These results are
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particularly important for developing countries where laboratories generally have
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scarce financial resources.
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ACKNOWLEDGMENTS
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We appreciate the contributions of Dra Maria L Abate, Dr Carlos E de Melo and Dr
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Antônio A Barone for the opportunity to complement our knowledge of the
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methodologies used. In addition, we wish to thank Geane L Flores, Islene Azevedo,
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Letícia P Scalioni, Moyra M Portilho and Selma XSL Pinheiro for technical assistance
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in collecting blood samples. Also, authors thank to Oswaldo Cruz Foundation
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(FIOCRUZ); FAPERJ and CNPq for financial support.
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Chu, T., Kulkarni, R., Gane, E.J., Roberts, S.K., Stedman, C., Angus, P.W. et al.,
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TABLES
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Table 1: Primers used in the tetra-primer ARMS-PCR and RFLP techniques
rs8099917
ARMS 860F1
CCA GGG CCC CTA ACC
(Forward outer)
TCT GCA CAG TCT G
ARMS 860R1
CTA TGT CAG CGC CCA
(Reverse outer)
CAA TTC CCA CCA C
ARMS 860F2T
ACT GAA CCA GGG ACG
(Forward inner)
TCC CCG AAG GAG T
ARMS 860R2C
CGG AGT GCA ATT CAA
(Reverse inner)
CCC TGG TGC G
ARMS 917F1
CAT CAC CTA TAA CTT
(Forward outer)
CAC CAT CCT CCT C
ARMS 917R1
GGT ATC AAC CCC ACC
(°C)
Amplicon
76.0
277 bp
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TCA AAT TAT CCT A
te
(Reverse outer)
Tm
cr
Primer Sequence (5’-3’)
M
rs12979860
Primer
d
SNP
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Tetra-primer ARMS-PCR
ARMS 917F2C
CTT TTG TTT TCC TTT
(Forward inner)
CTG TGA GCA GTG
ARMS 917R2T
TAT ACA GCA TGG TTC
(Reverse inner)
CAA TTT GGG TAA A
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76.0 75.0
198 bp
76.0
132 bp
65.0 437 bp 66.0 65.0
197 bp
66.0
295 bp
RFLP and Sequencing
SNP
Primer
rs12979860 860F (Forward)
860R (Reverse) rs8099917
917F (Forward)
Primer Sequence (5’-3’) GCT TAT CGC ATA CGG CTA GG AGG CTC AGG GTC AAT CAC AG TCA CCA TCC TCC TCT CAT CC
Tm (°C)
Amplicon
60.0 242 bp 60.0 60.0
539 bp
Page 13 of 18
ACC CTC TCC CCT TCC
917 R (Reverse)
TTT AG
59.0
286
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Table 2: Distribution of genotypes in 281 HCV chronic patients according to the
288
methods used in this study SNP rs12979860
SNP rs8099917
Genotypes
Genotypes
CT
TT
TT
Tetra-primer ARMS-PCR
80
135
66
173
RFLP
80
135
66
173
Real-time PCR
80
135
66
Sequencing
80
135
66
TG
GG
95
13
95
13
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CC
cr
Method
95
13
173
95
13
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173
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Figure 1: (A) A 2.5% agarose gel showing ARMS PCR profiles for SNP rs12979860
292
(CC, TT and CT genotypes). (B) A 2.5% agarose gel showing ARMS PCR profiles for
293
SNP rs8099917 (TT, TG and GG genotypes). (C) A 3% agarose gel showing RFLP
294
profiles for SNP rs12979860 (CC, CT and TT genotypes). (D) A 3% agarose gel
295
showing RFLP profiles for SNP rs8099917 (TT, TG and GG genotypes). Molecular
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Weight 100bp (Promega).
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HIGHLIGHTS
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Four methods used to detect IL28B SNPs in patients infected with HCV were compared.
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The methods were specific for genotyping the SNPs rs12979860 and
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rs8099917 of the IL28B gene.
ARMS-PCR method showed the best results in terms of cost-benefit analysis.
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ARMS-PCR method is easy to use as part of routine molecular diagnosis, with
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minimum equipment requirements.
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S0166-0934(15)00130-5 http://dx.doi.org/doi:10.1016/j.jviromet.2015.04.001 VIRMET 12765
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
26-11-2014 1-4-2015 2-4-2015
Please cite this article as: http://dx.doi.org/10.1016/j.jviromet.2015.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
COMPARISON OF FOUR METHODS OF GENOTYPING IL28B POLYMORPHISMS
2
IN CHRONIC HEPATITIS C PATIENTS
3
Nathália Delvauxa, Vanessa Duarte da Costaa, Maristella Matos da Costaa, Elisabeth
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Lampea
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Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, RJ, Brazil
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*Corresponding author: Nathália Delvaux, Laboratório Nacional de Hepatites Virais,
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Instituto Oswaldo Cruz - FIOCRUZ. Avenida Brasil, 4365 - Manguinhos - Código
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Postal: 21040-900. Rio de Janeiro, RJ, Brazil. Telephone: (+55) 21 2562 - 1894. E-
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mails:
and
[email protected]
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SUMMARY
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Background: Single nucleotide polymorphisms (SNPs) of the interleukin 28B
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(IL28B) gene are associated with viral clearance and treatment response in hepatitis
17
C virus (HCV) infection; however, most of the available SNP genotyping methods are
18
expensive.
19
Aims: This study sought to evaluate the cost effectiveness of four methods used to
20
genotype the rs12979860 and rs8099917 SNPs of the IL28B gene.
21
Methods: Tetra-primer amplification-refractory mutation system-polymerase chain
22
reaction (ARMS-PCR), restriction fragment length polymorphism (RFLP), quantitative
23
(q) PCR and direct sequencing methods were evaluated in terms of specificity, cost
24
and run time in 281 blood samples obtained from chronic HCV patients.
25
Results: In ARMS-PCR method, the primers designed to target both SNPs produced
26
PCR fragments of specific sizes that distinguished the alleles of rs12979860 and
27
rs8099917. In RFLP, the band profile allowed the distinction between genotypes. The
28
qPCR was the faster and easier to perform. Validation by nucleotide sequencing
29
showed 100% agreement among the three methods. The cost for a single reaction
30
was lowest for ARMS-PCR, followed in turn by RFLP, qPCR and sequencing.
31
Conclusions: The methodology described for the ARMS-PCR showed the most
32
favorable cost-benefit ratio. Moreover, this approach is fast and simple, requiring only
33
equipment that is commonly used in molecular diagnosis, which is an essential
34
parameter for use in developing countries where laboratories have scarce financial
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resources.
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Key
words:
IL28B,
SNP,
HCV,
Brazil,
ARMS-PCR
Page 2 of 18
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The treatment of chronic hepatitis C virus (HCV) infection, despite recent
40
progress following the introduction of direct-acting antiviral (DAA) regimens, remains
41
a great challenge in terms of cost effectiveness. Thus, the rapid identification of
42
sustained virologic response (SVR) predictors remains a major target in HCV
43
research. Two single nucleotide polymorphisms (SNPs) rs12979860 and rs8099917,
44
in close proximity to the interleukin 28B (IL28B) gene, have reported associations
45
with viral clearance and treatment response to HCV infection (Ge, et al., 2009;
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Tanaka et al., 2009). The IL28B gene encodes interleukin 28, a cytokine belonging to
47
the IFN-λ family that is involved in the regulation of the immune response against
48
viral infections (Dellgren et al., 2009; Li et al., 2009). The SVR rate with PEG-
49
IFN/RBV therapy in HCV-1 infected patients is two-fold higher in individuals with the
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rs12979860 CC genotype than the CT or TT (70-80% vs. 30-40%) (Chen et al., 2011;
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Thompson et al., 2010). Similarly, the SVR is higher in individuals with the rs8099917
52
TT genotype compared to the TT or GT+GG genotype (81% vs. 59%) (Sakamoto et
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al., 2011). Several clinical studies have validated these findings and, IL28B
54
genotyping has become an important tool to assist in clinical decisions regarding the
55
most appropriate therapeutic regimen. Furthermore, IL28B genotyping has shown
56
that it may be useful as a first-generation DAA approach for identifying patients who
57
can be treated successfully with a shorter and simpler treatment scheme (Jacobson
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et al., 2011; Poordad et al., 2011). Additionally, the impact of the IL28B CC genotype
59
(rs12979860) was observed in HCV-1a infected patients undergoing IFN-free
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combination therapy (58 to 84% among patients with IL28B CC vs. 33 to 64% in
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patients with non-CC genotypes) (Chu et al., 2011; Zeuzem et al., 2012, 2013).
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IL28B genotyping can be determined using diverse methods, such as DNA
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sequencing, Taqman assays, PCR-RFLP, and DNA high-performance liquid
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Chromatography (DHPLC) (Fiorina et al., 2012; Medrano and Oliveira, 2014). These
65
methods have different characteristics, including equipment needs, cost and
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technical knowledge. However, some of the methods are excessively expensive, and
67
their high cost of implementation limits their use, especially in developing countries
68
(Ferreira et al., 2013; Galmozzi et al., 2011; Medrano and Oliveira, 2014). Aiming to
69
obtain a cheap and simple test to determine IL28B genotype, in house protocols for
70
tetra-primer amplification-refractory mutation system-polymerase chain reaction
71
(ARMS-PCR) and for restriction fragment length polymorphism (RFLP) were
72
optimized in this study. Commercial qPCR method was also used and direct
73
sequencing was used to validate these techniques. Additionally, the four methods
74
used to determining the IL28B polymorphisms (rs12979860 and rs8099917) in
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patients with HCV infection were evaluated in terms of specificity, cost and run time.
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ARMS-PCR, RFLP, quantitative (q) PCR and direct sequencing methods were
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carried out in 281 blood samples (108 males, aged 56.0 ± 10.9 years) obtained from
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chronic HCV patients (positive anti-HCV antibody and detectable HCV RNA in serum
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samples of patients with infection for more than 6 months). The local Ethical
80
Committee (CEP Nº 297.459) approved this study. Genomic DNA was extracted from
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200 µL of whole blood using the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany),
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according to the manufacturer’s directions, and stored at -20°C.
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In ARMS-PCR assay, to design the primers to target the two SNPs, we used the
84
program
developed
by
Ye
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http://primer1.soton.ac.uk/primer1.html. The limiting of fragment sizes was chosen
86
within the range of 100–300 bp with a ratio of allelic bands of 1.5. Default settings
et
al.
(2001),
available
at
Page 4 of 18
were used for the other parameters. For rs12979860, the forward primer (FIT 860)
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was designed to hybridize to the genomic sequence with the T allele, and the reverse
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primer (RIC 860) was designed to hybridize with sequences containing the C allele.
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The same procedure was conducted for rs8099917 (FIG 917 inner - G allele, RIT 917
91
inner - T allele). The primers designed for ARMS-PCR assay are showed in table 1.
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PCRs for both SNPs were performed in a volume of 20 µL. Different amplification
93
conditions (annealing temperatures, PCR cycle protocols and primer concentrations)
94
were assessed to ensure proper formation of all the fragments. For rs12979860 the
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most appropriated PCR conditions were: 95°C for 15 min followed by 35 cycles of
96
94°C for 30 s, 64°C for 30 s, 72°C for 1 min and 72°C for 10 min. For rs8099917, the
97
best results were obtained with annealing temperature of 55°C using the same
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cycling conditions. The PCR products were separated by standard electrophoresis on
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2.5% agarose gels containing gel red dye (Biotium Inc., Hayward, CA), with a 100 bp
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molecular weight marker (Promega, Madison, Wisconsin, USA).
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The amplification results showed that the ARMS-PCR method distinguishes
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successfully between the two different SNPs. In the rs12979860 genotyping method,
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the outer primer pairs produced a band of 277 bp. The homozygote TT could be
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distinguished by an additional band of 198 bp, the homozygote CC could be
105
distinguished by a band of 132 bp and heterozygote C and T alleles produced
106
fragments of 132 bp and 198 bp, respectively. The genotypes of rs8099917 could be
107
differentiated according to the following band profiles: TT - 437 bp and 295 bp; GG -
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437 bp and 197 bp; and TG - 437 bp, 295 bp and 197 bp. Figure 1A shows an
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agarose gel representing ARMS-PCR profiles for SNP rs12979860, and Figure 1B
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shows the results for SNP rs8099917.
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For RFLP, two pairs of primers were designed for each IL28B gene SNP (Table
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1). The primer pairs 860F/860R were designed to amplify a fragment of 242 bp for
113
the rs12979860, and the primer pairs 917F/917R were designed to amplify a
114
fragment of 539 bp for rs8099917. The PCRs were performed in separate tubes
115
containing 0.2 pmol of the respective primer pairs. For rs12979860, the PCR
116
conditions were as follows: 95°C for 15 min, followed by 35 cycles of 95°C for 1 min,
117
58°C for 45 s, 72°C for 45 s and 72°C for 3 min. Amplified PCR products were
118
digested with the restriction enzyme BstUI (10 U/rxn; New England Biolabs) at 37°C
119
for 3.5 h. For rs8099917, PCR was conducted as follows: 95°C for 15 min; 10 cycles
120
of 95°C for 1 min, 50°C for 45 s, and 72°C for 45 s; and 25 cycles of 94°C for 30 s,
121
55°C for 30 s, 72°C for 30 s and 72°C for 3 min. The amplified PCR products were
122
digested with the restriction enzyme Tsp45I (2 U/rxn; New England Biolabs) at 65°C
123
for 3.5 h. The digested products of both SNPs were separated by standard
124
electrophoresis on 3% agarose gels containing gel red dye (Biotium Inc., Hayward,
125
CA), alongside 100 bp Molecular Weight (Promega, Madison, Wisconsin, USA). The
126
242 bp PCR fragment obtained with the primers designed for typing rs12979860
127
digested with BstUI produced the following band profiles: 3 fragments of 25 bp, 82 bp
128
and 135 bp in individuals with the CC genotype; 2 fragments of 82 bp and 160 bp in
129
individuals with the TT genotype; and 4 fragments of 25 bp, 82 bp, 135 bp and 160
130
bp in individuals with the heterozygote CT genotype (Figure 1C). The enzymatic
131
digestion of the 539 bp rs8099917 fragment with Tsp45I, produced a band profiles
132
that allowed the distinction between the GG (39 bp, 214 bp and 286 bp), TG (39 bp,
133
214 bp, 286 bp and 325 bp) and TT (214 bp and 325 bp) genotypes (Figure 1D).
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qPCR was performed using a commercial kit from Roche Life Technologies (TIB
135
MOLBIOL GmbH, Berlin, Germany), according to the manufacturer’s instructions.
Page 6 of 18
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The genotyping results for rs12979860 and rs8099917 with the qPCR method could
137
distinguish between the respective SNP genotypes according the fluorescence
138
profile. Direct sequencing after PCR amplification was done with the same primers as
140
those used for the RFLP for each polymorphism. The PCR products were purified
141
using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) and submitted to
142
nucleotide sequencing reactions in both directions using the Big Dye Terminator kit
143
3.1 (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s
144
instructions, followed by analysis on the ABI 3730 DNA automated sequencer
145
(Applied Biosystems, Foster City, CA, USA). In sequencing chromatogram,
146
sequences that showed single peaks were considered as homozygous, whereas
147
those that showed double overlapping peaks were interpreted as heterozygous. All
148
results obtained agreed 100% with the other three methods used in this study. The
149
distribution of genotypes according to the methods used in this study is shown in
150
Table 2.
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To estimate the cost of each technique, the price of reagents (enzymes, buffers,
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agarose, dyes, and other specific reagents for each procedure) and disposable
153
materials (tips, polystyrene tubes, gloves) used in each of the methods was
154
calculated based on currently available commercial prices. Other indirect costs were
155
not included, such as equipment maintenance and human resources. Thus, the price
156
of a single reaction for ARMS-PCR, RFLP, qPCR and direct sequencing was
157
respectively: US$19.40; US$27.80; US$22.90 and US$202.80. The assay that
158
requires more execution time was direct sequencing (2520 minutes or more),
159
followed by RFLP (570 minutes), ARMS-PCR (300 minutes) and commercial qPCR
160
(130 minutes).
Page 7 of 18
ARMS-PCR methodology showed the lowest cost and easiest execution, as well
162
as the second fastest run time. In addition, this method presents a wide convenience
163
of execution; it requires only equipment that is routinely used in most laboratories
164
that perform molecular biology assessments, such as a thermal cycler and
165
electrophoresis apparatus. In this technique, the high specificity of the reaction relies
166
on the 3’ terminus mismatch and the position-2 (second to the terminal) mismatch
167
from the 3’ terminus of the same allele-specific primer. This last mismatch
168
destabilizes the base paring between the primers and their corresponding non-target
169
templates and increases the specificity of the reaction by eliminating false-positive
170
results (Ye et al., 2001). This method uses four primers in a single PCR reaction; two
171
non-allele-specific primers (outer primers) amplify the region that comprises the SNP,
172
and as the outer primer fragment is produced, it serves as a template for the two
173
allele-specific primers (inner primers) that generate allele-specific fragments. By
174
placing the outer primers at different distances from the SNP, the two allele-specific
175
fragments can be distinguished by their different sizes in an agarose gel (Medrano
176
and de Oliveira, 2014; Ye et al., 2001).
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The RFLP technique was the second most expensive method and required a
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longer run-time. This method also requires two stages: PCR reaction and incubation
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with restriction enzymes. Furthermore, it requires two electrophoresis runs: one for
180
analysis of the PCR amplicons and the other for analyzing the band profiles after
181
enzymatic digestion that takes more than 3 h. In addition, restriction enzymes are
182
high in cost and are extremely unstable. On the other hand, despite its higher cost,
183
the equipments required for RFLP are routinely used in most molecular biology
184
laboratories (thermal cycler, electrophoresis apparatus and water bath) and the
185
training for this technique and interpretation of the results is not complex. Thus, even
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with disadvantages in several issues, the RFLP technique is, along with ARMS-PCR,
187
very accessible to laboratories with few financial resources. Among the four methods evaluated in this study, qPCR was the fastest and
189
easiest to perform. Moreover, this method is sensitive and specific due to Taqman
190
probe technology. qPCR was the second least expensive technique, even though it
191
uses a commercial kit. However, this method requires a specific and expensive
192
thermal cycler that is not available in many laboratories and requires additional
193
training for the interpretation and analysis of data.
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Direct sequencing is considered the gold standard and was used in this study to
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validate the results obtained with the other techniques. This method is the most time-
196
consuming and laborious of the procedures evaluated. The number of steps required
197
is large, including two PCR rounds, agarose gel electrophoresis, purification of PCR
198
products, and diverse handling steps, until the sequence is obtained as a
199
chromatogram. Also, it is necessary to have good knowledge of sequencing
200
programs to analyze the data, especially in the heterozygous case. Moreover, the
201
equipment is extremely expensive, and the maintenance cost is high because it
202
demands several specific reagents. In the present work, the results of all three
203
methods were 100% concordant with the results of direct sequencing.
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In conclusion, all methods tested were specific for genotyping SNPs rs12979860
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and rs8099917 of the IL28B gene. However, ARMS-PCR showed the best results
206
according to the cost-benefit analysis. This approach represents a simple, fast and
207
cost-effective method that involves a single PCR reaction followed by gel
208
electrophoresis. Therefore, this technique is easy to use in a routine molecular
209
diagnostic setting, with minimum equipment requirements. These results are
Page 9 of 18
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particularly important for developing countries where laboratories generally have
211
scarce financial resources.
212
ACKNOWLEDGMENTS
214
We appreciate the contributions of Dra Maria L Abate, Dr Carlos E de Melo and Dr
215
Antônio A Barone for the opportunity to complement our knowledge of the
216
methodologies used. In addition, we wish to thank Geane L Flores, Islene Azevedo,
217
Letícia P Scalioni, Moyra M Portilho and Selma XSL Pinheiro for technical assistance
218
in collecting blood samples. Also, authors thank to Oswaldo Cruz Foundation
219
(FIOCRUZ); FAPERJ and CNPq for financial support.
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REFERENCES
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IL28B genetic variations are associated with high sustained virological response
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(SVR) of interferon-α plusribavirin therapy in Taiwanese chronic HCV infection.
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Genes Immun. 12 (4), 300-309. doi:10.1038/gene.2011.1.
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Chu, T., Kulkarni, R., Gane, E.J., Roberts, S.K., Stedman, C., Angus, P.W. et al.,
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2011. The effect of host IL28B genotype on early viral kinetics during interferon-free
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treatment in patients with chronic hepatitis C. J. Hepatol. 54 (S1), S521.
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doi:10.1053/j.gastro.2011.12.057.
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Dellgren, C., Gad, H.H., Hamming, O.J., Melchjorsen, J., Hartmann, R., 2009.
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Ferreira, C.daS., Abreu, R.M., da Silva, M.C., Ferreira, A.S., Nasser, P.D.,
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Carrilho, F.J. et al., 2013. A fast and cost-effective method for identifying a
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polymorphism of interleukin 28B related to hepatitis C. PLOS ONE 8 (10), e78142.
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doi:10.1371/journal.pone.0078142. Fiorina, L., Paolucci, S., Papadimitriou, S., Baldanti, F., 2012. Comparison of
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Hepat. 18 (9), 628-630. doi:10.1111/j.1365-2893.2010.01349.x.
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Ge, D., Fellay, J., Thompson, A.J., Simon, J.S., Shianna, K.V., Urban, T.J. et al.,
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clearance. Nature 461 (7262), 399-401. doi:10.1038/nature08309.
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Jacobson, I.M., McHutchison, J.G., Dusheiko, G., Di Bisceglie, A.M., Reddy,
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K.R., Bzowej, N.H. et al., 2011. Telaprevir for previously untreated chronic hepatitis C
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virus infection. N. Engl. J. Med. 364 (25), 2405–2416. doi:10.1056/NEJMoa1012912.
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Li, M., Liu, X., Zhou, Y., Su, S.B., 2009. Interferon-lambdas: the modulators of
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Medrano, R.F., de Oliveira, C.A., 2014. Guidelines for the tetra-primer ARMS-
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PCR technique development. Mol. Biotechnol. 56 (7), 599-608. doi:10.1007/s12033-
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014-9734-4.
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Poordad, F., McCone, J. Jr, Bacon, B.R., Bruno, S., Manns, M.P., Sulkowski,
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M.S. et al., 2011. Boceprevir for untreated chronic HCV genotype 1 infection. N.
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Engl. J. Med. 364 (13), 1195–1206. doi:10.1056/NEJMoa1010494.
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Sakamoto, N., Nakagawa, M., Tanaka, Y., Sekine-Osajima, Y., Ueyama, M.,
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Kurosaki, M. et al., 2011. Association of IL28B variants with response to pegylated-
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interferon alpha plus ribavirin combination therapy reveals intersubgenotypic
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differences between genotypes 2a and 2b.J. Med. Virol. 83 (5), 871-878.
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Zeuzem, S., Soriano, V., Asselah, T., Bronowicki, J.P., Lohse, A.W., Müllhaupt,
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B. et al., 2013. Faldaprevir and deleobuvir for HCV genotype 1 infection. N. Engl. J.
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Zeuzem, S., Soriano, V., Asselah, T., Bronowicki, J.P., Lohse, A.W., Müllhaupt,
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B. et al., 2012. SVR4 and SVR12 with an interferon-free regimen of BI 201335 and
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BI 207127, +/- ribavirin, in treatment-naive patients with chronic genotype-1 HCV
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infection: interim results of SOUND-C2. J. Hepatol. 56 (Suppl. 2), S45.
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TABLES
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Table 1: Primers used in the tetra-primer ARMS-PCR and RFLP techniques
rs8099917
ARMS 860F1
CCA GGG CCC CTA ACC
(Forward outer)
TCT GCA CAG TCT G
ARMS 860R1
CTA TGT CAG CGC CCA
(Reverse outer)
CAA TTC CCA CCA C
ARMS 860F2T
ACT GAA CCA GGG ACG
(Forward inner)
TCC CCG AAG GAG T
ARMS 860R2C
CGG AGT GCA ATT CAA
(Reverse inner)
CCC TGG TGC G
ARMS 917F1
CAT CAC CTA TAA CTT
(Forward outer)
CAC CAT CCT CCT C
ARMS 917R1
GGT ATC AAC CCC ACC
(°C)
Amplicon
76.0
277 bp
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TCA AAT TAT CCT A
te
(Reverse outer)
Tm
cr
Primer Sequence (5’-3’)
M
rs12979860
Primer
d
SNP
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Tetra-primer ARMS-PCR
ARMS 917F2C
CTT TTG TTT TCC TTT
(Forward inner)
CTG TGA GCA GTG
ARMS 917R2T
TAT ACA GCA TGG TTC
(Reverse inner)
CAA TTT GGG TAA A
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76.0 75.0
198 bp
76.0
132 bp
65.0 437 bp 66.0 65.0
197 bp
66.0
295 bp
RFLP and Sequencing
SNP
Primer
rs12979860 860F (Forward)
860R (Reverse) rs8099917
917F (Forward)
Primer Sequence (5’-3’) GCT TAT CGC ATA CGG CTA GG AGG CTC AGG GTC AAT CAC AG TCA CCA TCC TCC TCT CAT CC
Tm (°C)
Amplicon
60.0 242 bp 60.0 60.0
539 bp
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ACC CTC TCC CCT TCC
917 R (Reverse)
TTT AG
59.0
286
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Table 2: Distribution of genotypes in 281 HCV chronic patients according to the
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methods used in this study SNP rs12979860
SNP rs8099917
Genotypes
Genotypes
CT
TT
TT
Tetra-primer ARMS-PCR
80
135
66
173
RFLP
80
135
66
173
Real-time PCR
80
135
66
Sequencing
80
135
66
TG
GG
95
13
95
13
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CC
cr
Method
95
13
173
95
13
us
173
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Figure 1: (A) A 2.5% agarose gel showing ARMS PCR profiles for SNP rs12979860
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(CC, TT and CT genotypes). (B) A 2.5% agarose gel showing ARMS PCR profiles for
293
SNP rs8099917 (TT, TG and GG genotypes). (C) A 3% agarose gel showing RFLP
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profiles for SNP rs12979860 (CC, CT and TT genotypes). (D) A 3% agarose gel
295
showing RFLP profiles for SNP rs8099917 (TT, TG and GG genotypes). Molecular
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Weight 100bp (Promega).
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HIGHLIGHTS
298
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Four methods used to detect IL28B SNPs in patients infected with HCV were compared.
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301
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The methods were specific for genotyping the SNPs rs12979860 and
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rs8099917 of the IL28B gene.
ARMS-PCR method showed the best results in terms of cost-benefit analysis.
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304
306
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ARMS-PCR method is easy to use as part of routine molecular diagnosis, with
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minimum equipment requirements.
312 313
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These results are important for developing countries where laboratories typically have scarce financial resources.
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Figure(s)
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