Absolute Quantitation of Different Genotypes of Hepatitis C Virus RNA in Clinical Samples by a Modified Real-time PCR Method

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Absolute Quantitation of Different Genotypes of Hepatitis C Virus RNA in Clinical Samples by a Modified Real-time PCR Method

Abstract

Background: Our aim was to develop a quantitative real-time polymerase chain reaction (qPCR) assay for the quantitation of hepatitis C virus (HCV) RNA samples from patients infected with different HCV genotypes.Methods: A standard curve was generated by amplification of serial dilutions of HCV-plasmid (pFKI389-NS3-3') harboring genotype 1b HCV-subgenomic replicon. Samples from 15 HCV-infected patients (genotypes 1, 2, and 3) were analyzed to quantify HCV-RNA by qPCR with primers and probes specific for the 5'-UTR viral region.Results: The HCV qPCR assay had a sensitivity of 100 copies/reaction with a dynamic range of detection between 102–20 × 106 HCV copies. The assay was highly reproducible with a low coefficient of variation. We observed that the HCV genotypes included could be identified by our method.Conclusions: Our results showed that this modified qPCR assay provides

a valid platform for quantifying HCV-RNA, combining good analytical sensitivity with a wide dynamic range and high reproducibility.

Introduction

Hepatitis C virus (HCV) infection is a major global health problem that is growing and affecting about 170 million people worldwide.[1–4] Current standard treatment for chronic hepatitis C is the combination of pegylated interferon alpha (PEG-IFN-α) 2a or 2b and ribavirin,[1] which leads to a successful outcome in only about 50%-60% of individuals.[2] A sustained virological response (SVR) is defined as the absence of detectable HCV-RNA in blood 24 weeks after the completion of antiviral treatment.[3,4]

The demand for predicting the response to interferon-based treatment and monitoring its therapeutic efficacy have led to the development of a variety of methods to quantify the viral load in HCV-infected patients.[4–6] Most HCV quantification methods available are based on real-time reverse transcription-polymerase chain reaction (qPCR). The National Institute for Biological Standards and Controls and the World Health Organization certified a uniform standard for measuring HCV-RNA (based on genotype 1 panels) in international units (IU).7 To date, the following 2 qPCR assays are available: the Abbott RealTime HCV assay (ART) (Abbott Molecular, Des Plaines, IL) and the Roche Cobas AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM) (Roche Molecular Systems, Pleasanton, CA), but little is known about the variation of these assays for quantification of

different HCV genotypes in clinical samples.[7–9] It has been reported that approximately 15% of HCV genotype 2 and 30% of HCV genotype 4 samples are substantially underestimated with the CAP/CTM, whereas this problem has not been found with the ART assay.[10,11] We developed a modified, highly specific, sensitive, and reproducible reverse transcription polymerase chain reaction (RT-PCR) method allowing quantification of HCV-RNA in serum samples. We also examined the validity of this method in serum and plasma samples from patients infected with different HCV genotypes.

Clinical Samples

The sensitivity, specificity, linearity, and reproducibility of the developed method were evaluated using a synthetic template and clinical samples. We included 15 HCV-infected patients (4 men and 11 women with a mean age of 47 ± 13 years [range 22–60]) enrolled in the Liver Unit at Dr. José E. González University Hospital. No patient had received antiviral treatment at the time of the investigation. In addition, we included samples from 5 healthy voluntary blood donors. The research protocol was reviewed and approved by the institutional review board. At enrollment, all participants signed a written informed consent.

Serological Analysis

Chronic HCV infection was diagnosed by demonstrating the simultaneous presence of anti-HCV antibodies and HCV-RNA by qualitative RT-PCR. Anti-HCV detection was carried out with a third-generation enzyme immunoassay according to the manufacturer's instructions (Abbott Laboratories, Abbott Park, IL).

RNA Isolation

RNA was extracted from 1 milliliter of serum or plasma using a Qiagen Viral RNA Kit (Hilden, Germany) according to the manufacturer's instructions. The isolated RNA was resuspended in 60 μL of diethylpyrocarbonate (DEPC)-treated water and stored at -70°C until molecular analysis.

Synthesis of cDNA and Qualitative Detection of HCV-RNA

The isolated RNA was subjected to reverse transcription using a high-capacity cDNA Archive Kit (Applied Biosystems, City, CA) according to the manufacturer's specifications. The resultant cDNAs were subsequently used for conventional PCR and qPCR assay. For the qualitative detection of HCV-RNA by nested PCR, the primers were designed to amplify a portion of 5' untranslated region of HCV genome. The primer pair for the first round of PCR was: sense-primer HCV-U1 5'-CTGTGAGGAACTACTGTCTTC-3' and antisense primer HCV-D2 5'-CAACACTACTCGGCTAGCAGT-3'. For the second round a set of sense-primer HCV1-N3 5'-ACGCAGAAAGCGTCTAGCCAT-3' and antisense primer HCV-N4 5'-ACTCGGCTAGCAGTCTTGCGG-3' were used. The PCR reagents and thermal cycling conditions were used according to the protocol previously described.[12]

Genotyping Analysis

We used the reverse hybridization Inno-Lipa HCV II assay (Innogenetics-Bayer, Antwerp, Belgium) according to the manufacturer's instructions to identify HCV genotypes. The HCV genotype nomenclature used in this study is that proposed recently by an international panel.[13]

Preparation of the Standard Curve

To perform an absolute quantitative qPCR, a plasmid construct (pFKI389-NS3–3') harboring genotype 1b HCV subgenomic replicon[14] was used to prepare the standard curve. We first calculated the mass of a single plasmid molecule based on the pFKI389-NS3-3' plasmid size, the mass of plasmid containing the copy number of interest that was 20,000,000 to 10 copies, and the concentration of plasmid needed to achieve the copy number of interest. This was followed by the preparation of 10-fold serial dilutions of plasmid template to create the standard curve. Finally, with the threshold cycle (Ct) values, a linear regression model was calculated.

Absolute Quantitative PCR Assay

cDNAs (5μL) were subjected to qPCR for HCV-RNA quantification. Amplifications were conducted in triplicate using the following primers: HCV-Forward (+75–93 nt) 5'-GCGTCTAGCCATGGCGTTA-3' and HCV-Reverse (+138–157 nt) 5'-GGTTCCGCAGACCACTATGG-3'; and the TaqMan probe (+94–110 nt) 5'-FAM-CTGCACGACACTCATAC-NFQ-3'. The Perkin-Elmer ABI Prism 7000 Sequence Detection System was used for real-time analysis and thermal cycling conditions were: initial setup at 50°C for 2 minutes, then 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Fluorescence was monitored during each annealing step and amplification plots were generated. For each PCR reaction, 12.5 μL of TaqMan PCR Master Mix, 1.25 μL of 20x Assay Mix, and 11.25 μL of cDNA diluted were added.

Variations between replicates and runs are expressed as means, standard deviations (SD), and coefficients of variation (CV). Moreover, the coefficient of determination (R 2) was calculated. Statistical significance was set at P<0.05.

Sensitivity and Linearity of the Real-time PCR Method

The analytical sensitivity of our method was determined by serial dilutions of pFKI389-NS3-3' HCV subgenomic replicon plasmid containing from 0 to 20 × 106 copies tested 5 times; each standard dilution was tested in triplicate. The detection limit was determined at 100 HCV copies per reaction with 100% detection ability (3 of 3 replicates). The correlation coefficient ® was 0.99 (Figure 1).

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Figure 1.

Linearity of real-time HCV quantification assay. Linearity was determined using serial 10-fold dilutions of pFKI389-NS3-3' HCV plasmid. Linear relationship between the Ct values and the log10 plasmid copy number yielded r=0.99.

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Figure 1.

Linearity of real-time HCV quantification assay. Linearity was determined using serial 10-fold dilutions of pFKI389-NS3-3' HCV plasmid. Linear relationship between the Ct values and the log10 plasmid copy number yielded r=0.99.

Reproducibility and Specificity of the Real-time PCR

In order to determine the reproducibility of intra- and inter-measurements of the qPCR, serial dilutions were tested on 2 separated days (interassay) with triplicates of each dilution in each run (intrassay). Table 1 shows the mean CV of Ct values and the input copy number of HCV within a run; the CV calculated for these repetitions was less than 1.06%. Interassay CV was low for standard curve and samples (Table 1), a reasonable target for the percentage of CV in routine testing is 10%-15%. On the other hand, the reproducibility of the RT-PCR was determined using 4 samples from patients chronically infected with HCV genotypes 1, 2, and 3; the most frequent genotypes in Northeast Mexico.[20,23] Table 1 shows detection of all HCV subtypes tested. The CV of these assays was less than 0.45%, and each sample was tested in triplicate.

The specificity of the RT-PCR we developed was assessed by testing serum/plasma samples from 5 healthy blood donors. All of these samples were negative for HCV-RNA.

Quantitation of HCV-RNA in Clinical Samples

The HCV-RNA was quantified in 15 samples from HCV-infected patients. The results obtained with our assay were compared with results from the AMPLICOR HCV Monitor Assay, which was performed as recommended by the manufacturer. The conformity of the quantitative titers between both assays is shown in Figure 2. Quantitative titers ranged from 102 to 20 × 106 copies/mL (median, 1.8 × 105 copies/mL) as determined by in-house PCR, and from 2 × 102 to 23 × 106 copies/mL (median, 1.9 × 105 copies/mL) by AMPLICOR HCV Monitor Assay.

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Figure 2.

Correlation between the results for 15 HCV-infected patients tested by Amplicor HCV Monitor v2.0 Assay and modified qPCR, irrespective of the genotype. Results show a good correlation between the two assays (r=0.98).

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Figure 2.

Correlation between the results for 15 HCV-infected patients tested by Amplicor HCV Monitor v2.0 Assay and modified qPCR, irrespective of the genotype. Results show a good correlation between the two assays (r=0.98).

As shown in Table 2, all samples were quantified by the modified HCV qPCR method, and results are reported in copies/mL and IU/mL. Conversion factors established by international guidelines can be used to establish a relationship between the IUs and non-standardized copies. Because of this, we used IU conversion factors for the non-standardized units previously used in commercial HCV-RNA quantitative assays as follows (AMPLICOR HCV Monitor Assay, 1 IU/mL=0.9 copies/mL) (Table 2).[15]

In HCV-infected patients, the quantification of viral RNA has been regarded as 1 of the most important indicators for the outcome of interferon therapy.[2,4] Real-time PCR assays have become an essential tool to monitor viral load. Based on this, we developed a highly specific, sensitive, and reproducible real-time PCR method allowing the quantification of HCV-RNA in serum and plasma samples from patients infected with different genotypes.

In this study, the sensitivity, specificity, linearity, and reproducibility of an in-house HCV real-time PCR assay were evaluated. We found that the dynamic range of quantification of our method was from 102 to 20 × 106 copies/mL. The correlation coefficient ® was 0.99 (Figure 1). Kawai and colleagues16 reported that their TaqMan PCR assay had a sensitivity from 2 × 103 to 2 × 108 copies/mL. Hence, we were able to detect a low copy number compared with assays mentioned before. On the other hand, interassay and intrassay reproducibility were evaluated, and CV values were low (Table 1). It has been reported that a CV of <20% is considered adequate for the reproducibility of the assay.[16] We also

found that the reproducibility of our method using clinical samples had a CV less than 0.45%, and different HCV genotypes can be efficiently identified (Table 1). The specificity of our method was high as demonstrated by the absence of HCV-RNA in all samples from negative HCV controls.

In the 15 patients analyzed, we found the following distribution of HCV genotype: 1, 6.7%; 1a, 13.3%; 1b, 26.6%; 1a/1b, 13.3%; 2b, 20%; 3, 6.7%; 3a, 6.7%; and 3b, 6.7%. The genotype pattern was similar to those previously reported in the Northeast and other regions of Mexico.[17] We demonstrated that HCV-RNA levels measured by the AMPLICOR Monitor Assay correlated with those measured by the TaqMan PCR Assay (r=0.98) (Figure 2), and that it has a practical application and high specificity in samples from HCV-infected patients (Table 2).

In conclusion, our in-house real-time PCR method provides a valid platform for quantifying HCV-RNA, combining good analytical sensitivity with a wide dynamic range and high reproducibility. We suggest that our assay has a broad applicability in basic and clinical investigations requiring quantification of HCV-RNA levels.

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