A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

Hepatitis C virus (HCV) is one of the major causes of chronic liver diseases, which has infected an estimated 170 million people worldwide [1, 2]. It is responsible for chronic liver diseases and is a risk factor for liver cirrhosis and hepatocellular carcinoma [3]. Early diagnosis and evaluation of HCV cases is very helpful for the management of the disease.

Since enzyme immunoassays have been used for blood-donor screening and laboratory diagnosis of HCV infection, a sharp decline has been observed in post-transfusion hepatitis C [4‐6]. However, even with the most advanced third-generation assays, the HCV-antibody window period is approximately 58 days [7]. In addition, false-positive results may occur in patients with autoimmune diseases and in neonates born from mothers with chronic HCV infection [8‐10]. Screening of HCV RNA by using nucleic acid amplification techniques (NATs) reduces the risk of HCV transmission and aids in the early detection of HCV infections [11]. Recently, assays based on real-time reverse transcriptase-polymerase chain reaction (RT-PCR) have been introduced in routine diagnostics and are rapidly replacing assays based on standard RT-PCR and signal amplification [12]. Unlike serological assays, those based on real-time RT-PCR can be used for the diagnosis of acute hepatitis before seroconversion and in the case of some seronegative patients with immune deficiency. Detection based on real-time RT-PCR is also useful for confirming indeterminate serological results and monitoring response to treatment [13].

The HCV genome is extremely heterogeneous. The reason for this genetic heterogeneity is the high error rate due to the lack of proofreading ability of the RNA-dependent RNA polymerase, which is responsible for the replication of the viral genome. Published sequence data indicate that the 5' untranslated region (UTR) is generally highly conserved among different HCV isolates [14] and is the target of most HCV assays. This region, however, also contains genotypically variable sequence positions, which allow discrimination of all the major types and many subtypes of HCV [15]. Several researchers have confirmed that nucleotide mutations and polymorphisms exist in the 5' UTR of the HCV genome [16‐19]. Nucleic acid-based assays depend on hybridization between the template and PCR primers/probes [20], and mismatches can significantly reduce the viral detection and quantification efficiency. Thus, a single primer/probe, which is generally used in commercial HCV assays, may result in missing detections because of mismatches [12, 21, 22]. A duplex primer/probe assay can simultaneously amplify more than one target sequence [23, 24]. Theoretically, some specimens are likely to be missed out on testing with a singleplex primer/probe assay but are detected by a duplex primer/probe assay. Some researchers have proved this by the use of multiple primer/probe sets, which significantly improved the performance of nucleic acid-based assays [25, 26]. Sometimes, PCR inhibitors cannot be reliably removed from the sample and viral RNA may somewhat be degraded or may not be efficiently removed from the viral coat protein. Under these circumstances, internal controls (ICs), which are coextracted and coamplified with the viral RNA in the same reaction tube, can monitor the specimen extraction and amplification efficiency [27, 28]. Thus, false-negative results can be avoided with the use of ICs.

In this study, we developed a duplex real-time RT-PCR assay using 2 sets of primers/probes and a specific armored RNA as IC. With the combination of the 2 sets of primers/probes, the performance of the assay was significantly improved, avoiding missing detections to the maximum possible extent.

In this study, all the 5' UTR sequences of HCV recorded in the Los Alamos National Laboratory HCV Sequence Database were aligned. The alignment results revealed several nucleotide polymorphisms in the 5' UTR. Thus, all HCV sequences cannot be detected by a singleplex primer/probe assay. In order to avoid missing detections because of mismatch between the template and PCR primers/probes, the duplex primer/probe assay was used. In this assay, 2 probes were labelled with the same fluorophore (FAM) at the 5' end; these probes could mutually combine, greatly improving the power of the test (Figure 3).

Compared with a singleplex primer/probe set, the duplex primer/probe set has many advantages. First, the duplex primer/probe set could detect all the HCV genotypes in the HCV RNA Genotype Panel for NATs and avoided missing detections to the maximum possible extent. Several assays using a singleplex primer/probe set produce false-negative results because of mismatches between the template and primers/probes [12, 21, 22, 29]. This problem could be effectively resolved by using 2 sets of primers/probes, which has been proved in this study. The 2 sets of primers/probes could match interchangeably, improving the power of the test. For instance, Chevaliez et al. [30] reported the case of 2 patients infected with HCV genotype 4, whose serum samples with high viral load could not be detected by the CAP/CTM assay. Researchers found that the failing detections were probably related to nucleotide polymorphisms at positions 145 and 165. On the basis of the sequences of the 2 undetected HCV samples, we found that the 2 sets of primers/probes could detect these samples in theory, regardless of the nucleotide polymorphisms. Second, the duplex primer/probe assay can estimate the virus levels accurately. There are many reports about the underestimation of virus load by singleplex primer/probe assays [12, 21, 22, 29, 31]. For example, some patients with very low HCV viraemia may yield a negative result by the CAP/CTM assay [31]. The 2 sets of primers/probes used in our assay could match interchangeably, creating additional combinations with different primer-directed elongations. Figure 2 shows the performances of the duplex primer/probe and singleplex primer/probe assays in the testing of the same serum sample. Obviously, the fluorescence value of the 2 sets of primers/probes is higher than that of the single set of primers/probes, and cycle threshold (Ct) can shift towards left. As a result, the duplex primer/probe assay could strengthen the fluorescence signal of the low HCV viraemia samples and increase the probability of detection. Third, compared with two commercial HCV detection assays (BIOER and Kehua HCV fluorescence detection kits), the duplex primer/probe assay has many advantages. BIOER HCV fluorescence detection kit required 900-μl of serum for HCV RNA extraction. However, the duplex primer/probe assay barely needs 100-μl of serum for nucleic acid extraction. The latter has more wide range of application, especially in the case of fewness of sample. Moreover, the duplex primer/probe assay has lower LOD (38.99 IU/ml) than BIOER and Kehua HCV fluorescence detection kits, and the cost of the duplex real-time RT-PCR assay was lower than that of the two commercial HCV detection assays. Fourth, the sensitivity of the duplex primer/probe assay is high and can be compared with that of the CAP/CTM assay. In the CAP/CTM assay, HCV RNA was extracted from 850-μl serum and then eluted with 65-μl of elution buffer. Finally, 50-μl extract was used as the template in 100-μl reaction volume [32]. In the duplex primer/probe assay, HCV RNA was extracted from 100-μl serum and then eluted with 20-μl of diethyl pyrocarbonate-treated H₂O. Finally, 10-μl extract was used as the template in 25-μl reaction volume. The LOD of the duplex primer/probe assay was 38.99 IU/ml, which is higher than that of the CAP/CTM assay (15.0 IU/ml). Considering that HCV RNA was extracted from 850-μl serum and the reaction volume increased to 100-μl, we believed that the LOD of the duplex primer/probe assay could be comparable with or even exceed that of the CAP/CTM assay.

In this study, armored RNA was successfully used as IC in the duplex real-time RT-PCR assay. The IC spiked into the specimens could monitor the specimen extraction and amplification efficiency, saving additional labour-intensive procedures and expenditure of costly external control reagents. ICs include noncompetitive ICs and competitive ICs (CICs). In the noncompetitive IC strategy, separate primer pairs are used to detect ICs and the target nucleic acids. In a previous study, the performance of noncompetitive ICs was not perfect for the target nucleic acids because of differences in the amplification efficiencies [33]. In our study, CICs were constructed, which hybridized with the same primers and had identical amplification efficiencies as the target nucleic acid but contained discriminating probe-binding sequences [34]. In order to avoid the suppression of target amplification, the concentration of armored RNA spiked into the samples was optimized. According to the results shown in Table 1, 1000 copies/ml of armored RNA was used as the optimal concentration in the duplex real-time RT-PCR assay.

In the 109 serum samples collected from Shenzhen Blood Center, the prevalent genotypes of HCV should be 1 and 2 [35]. In the study of Chevaliez et al. [30], the CAP/CTM assay failed to detect HCV genotype 4. Thus, the testing results of the duplex real-time RT-PCR for the 109 serum samples, which were identical to those of the CAP/CTM assay, should be correct. The LOD of the duplex real-time RT-PCR assay was 38.99 IU/ml and the specificity was 100%. Furthermore, the cost of the duplex real-time RT-PCR assay was considerably lower than that of the CAP/CTM assay, and hence, the former assay is more suitable for large-scale use.

The duplex real-time RT-PCR assay is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.