Subgenotyping by one-step real-time PCR with MGB probe in the HCV NS5B region

In the present study, HCV genotypes of all samples were determined by the antibody serotyping method of Tukiyama-Kohara et al. [13,14]. According to this assay, HCV serotypes 1 and 2 correspond to HCV genotypes 1a/1b and 2a/2b [5]. The clinical background of 80 patients was shown in Table 1. All but 2 patients had high viral loads. First, all 80 patients were Japanese, and their HCV subgenotypes were determined by real-time PCR-based method with MGB probe in the HCV NS5B region [12]. HCV subgenotypes were determined in 74 of the 80 patients: 4 (5.0%) and 70 (87.5%) were classified into HCV subgenotypes 1a and 1b, respectively. HCV subgenotypes remained undetermined by this method in the other 6 patients (7.5%).

Comparison of the result from HCV subgenotyping by real-time PCR-based method in the HCV NS5B region and that from direct-sequencing by Sanger method in Japanese patients infected with HCV genotype 1

It has been reported that the prevalence of HCV subgenotype 1a was ~1% in Japan [11]. Our result from the real-time PCR-based subgenotyping method suggested that this prevalence might be a little higher although our study population of 80 patients was too small to be considered representative. We tried to perform direct-sequencing by the Sanger method in 10 patients: 4 with HCV subgenotype 1a and 6 with undetermined HCV subgenotypes by real-time PCR-based method [12]. Because the PCR product in 1 of the 4 HCV subgenotype 1a patients could not be amplified, this patient was excluded (Table 2, patient No. 69).

TaqMan MGB probes exhibit great differences in Tm values between matched and mismatched probes, providing more accurate allelic discrimination and making for a more sensitive real-time assay [12]. Thus, we then performed direct-sequencing by the Sanger method in 9 patients. As shown in Table 2, only 1 of three patients classified into HCV subgenotype 1a by real-time PCR-based method [12] was confirmed to be HCV subgenotype 1a (Table 2, patient No. 74). All other samples with subgenotype 1a or undetermined by real-time PCR-based method, were confirmed to be HCV subgenotype 1b by direct-sequencing (Table 2). In accordance with the alignments of the MGB probe segments from each patient in comparison to those in H77 and Con1, we found that patients No. 46 and 48 showed an A to G mutation at nucleotide site 8917 and 8918 (reference to Con1), respectively (Table 3).

Phylogenetic tree analysis.

We also constructed a phylogenetic tree based on the 9 patients’ sequences of about 424 bp length containing the HCV NS5B probe region obtained using direct-sequencing by the Sanger method. The phylogenetic tree showed that only one patient, No. 74, belonged to HCV subgenotype 1a, while the others all clustered to HCV subgenotype 1b (Figure 1).

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Figure 1. Phylogenetic tree analysis by the Neighbor-Joining (NJ) method.

The numbers at the branches are confidence values based on Felsenstein’s bootstrap analysis (500 replicates) with MEGA version 4 [28]. HCV HCV-J, HCV Con1 and HCV H77 strains belong to HCV subgenotypes 1b, 1b and 1a, respectively. GenBank accession No: HCV-J, D90208.1; Con1, AJ238799.1; H77, AF009606.1.

https://doi.org/10.1371/journal.pone.0073615.g001

Patients

Sera from 80 consecutive treatment-naïve chronic hepatitis C genotype 1 patients with well-characterized clinical follow-up were selected from a chronic hepatitis C database at Chiba University Hospital, Chiba, Japan. All patients were negative for hepatitis B and HIV antibodies. Sera from patients were stored at -20°C until analysis. This study was approved by the Ethics Committee, Chiba University, Graduate School of Medicine, Chiba, Japan (permission numbers 1462 and 282), and conformed to the Helsinki Declaration. Written informed consent was obtained from all patients before enrollment in this study.

RNA extraction

Total RNA was extracted from 140 µL of each serum sample using a QIAamp Viral RNA Mini Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. RNA was eluted in 60 µL of elution buffer and quantified using a NanoDrop Lite spectrophotometer (Thermo Scientific, Madison, WI, USA).

HCV subgenotyping by real-time PCR-based methods in the HCV NS5B region

Reactions were performed in a final volume of 50 µL using the Superscript™ III Platinum one-step quantitative RT-PCR system (Invitrogen, Carlsbad, CA, USA) as previously described [12]. The designed primers and MGB probes shown in Table 5 and the real-time PCR HCV subgenotyping protocol were used according to the previous description [12]. The reaction mixture was prepared in two fractions. The first fraction (F1) consisted of 18.5 µL of 2X reaction mix (6 mM MgSO₄, and 0.4 mM of each dNTP), 1.0 µL of Taq mix (Superscript™III RT, Platinum Taq Mix, Invitrogen), 200 nM of reverse primer (R56_1), and 0.5 µL of RNase Out (Invitrogen). Twenty-one microliters of F1 was added to optical tubes. The second reaction mix fraction (F2) consisted of 4.4 µL of 2X reaction mix, 200 nM of forward primer (F56_1), 1.5 µL of 50 mM MgSO₄, 0.1 µL of ROX (25 µM), and 200 nM of each probe (against either genotype 1a or 1b). A final F2 volume of 10 µL was transferred to another optical tube. After the addition of 19 µL of RNA, F1 was carefully added to F2. Reverse transcription was carried out in a TakaRa PCR Thermal Cycler (TaKaRa, Ohtsu, Shiga, Japan) at 55°C for 35 min with the thermocycler cover open. Following this, the tubes were briefly centrifuged and real-time PCR was carried out in a 7300 Real-Time PCR System (Applied Biosystems, Foster, CA, USA) using the following cycling parameters: 50°C for 2 min; 95°C for 10 min; and 40 cycles at 95°C for 15 sec, 50°C for 30 sec, and 60°C for 1 min. The total time required for performing this assay was only 2 hours, as previously described [12].

cDNA synthesis and amplification by PCR for ultra-deep sequencing

To perform ultra-deep sequencing of the MGB probe region in HCV NS5B, we used the HPLC-purified specific primers shown in Table 6. cDNA was synthesized with R56_1 (antisense) for 1 cycle at 55°C for 30 min and 85°C for 5 min using a Transcript high-fidelity cDNA synthesis kit (Roche, Tokyo, Japan). Then amplification was performed with Pr1 (sense) and R56_1 (antisense) for 35 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec using a FastStart high-fidelity PCR system, dNTPack kit (Roche).

Then, the first PCR product was further amplified with two inner primer sets using the FastStart high-fidelity PCR system, dNTPack kit (Roche). Set one was Pr2 (sense primer) and R56_1 (antisense primer), and the other was Pr1 (sense primer) and Pr2 (antisense primer). Considering the MGB probe locations at nt 8913-8926(1a) in reference sequence HCV subgenotype 1a, strain H77 (AF009606.1), and nt8910-8923(1b) in reference sequence HCV subgenotype 1b, strain Con1 (AJ238799.1), primer set one was used for PCR. The PCR conditions were as follows: 35 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec. Amplified products were separated by agarose gel electrophoresis and purified using a high pure PCR clean-up micro kit (Roche). Each amplicon was quantified using a NanoDrop Lite spectrophotometer (Thermo Scientific), and all amplicons from a single viral genome were pooled together at equimolar ratios. Each pool was then quantitated, and approximately 500 ng of each was used in a fragmentation reaction mix using a GS FLX Titanium Rapid Library Preparation Kit (Roche). Final libraries representing each genome were characterized for average size by using an Agilent High Sensitivity DNA kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Loveland, CO, USA). 4 x 10⁷ molecules of these final DNA libraries were then subjected to emulsion PCR, and enriched DNA beads were loaded onto a picotiter plate and pyrosequenced with a Roche/454 GS Junior sequencer using Titanium chemistry (454 Life Sciences Corp., Branfold, CT, USA). GS Amplicon Variant Analizer Version 2.7 (Roche) was used for read mapping and calculating variant frequencies at each nucleotide position according to reference sequence HCV subgenotype 1a, strain H77 or HCV subgenotype 1b, strain Con1.

Direct-sequencing by Sanger method

Then, we amplified the first PCR product using primer set one and TaKaRa Ex Taq (TaKaRa). The PCR conditions were as follows: 40 cycles at 98°C for 10 sec, 55°C for 30 sec, and 72°C for 60 sec; the last cycle followed at 72°C for 7 min. Sanger sequencing was performed using a BigDye(R) Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Tokyo, Japan). Sequences were detected using Applied Biosystems 3730xl. Nucleotide sequences were analyzed by GENETYX 10 (GENETYX Corp., Tokyo, Japan) and imported to MEGA version 4 [28], which was used to align the sequences, according to reference sequences.

Construction of phylogenetic trees

A fragment of about 424-bp length containing the HCV NS5B region was used for phylogenetic analysis. Phylogenetic trees were inferred using the Neighbor-Joining (NJ) model and robustness of the tree branches was tested using bootstrap analysis (500 replicates) with MEGA version 4 [28].

Nucleotide sequence accession number

All sequence reads have been deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive under accession number DRA001077.

Statistical analysis

Data were expressed as mean + standard deviation. We used univariative analyses, applying Student’s t-test or Chi-square test as appropriate. P 0.05 was considered statistically significant.