A dilution series of the World Health Organization (WHO) Second International Standard for HCV RNA (National Institute for Biological Standards and Control (NIBSC), code 96/798, UK) was used to determine the limit of detection (LOD) of the duplex real-time RT-PCR assay at the following concentrations: 10, 25, 50, 102, 103, 104, and 105 IU/ml. Each dilution of the WHO Standard was tested in a batch of 4 replicates in 6 separate runs, i.e. for each dilution, a total of 24 replicates were tested.
Linearity of the duplex real-time RT-PCR assay was determined using serial 10-fold dilutions of a clinical sample at the following concentrations: 10, 102, 103, 104, 105, and 106 IU/ml. At each concentration, 3 replicates were tested in a single run.
Inter-assay and intra-assay variations were calculated using a set of 3 samples with different viral loads (105, 104, and 102 IU/ml), which were tested 10 times in 3 different assays on different days.
The HCV RNA Genotype Panel for NATs (NIBSC, code 02/202, UK) was used to assess the performance of the duplex real-time RT-PCR assay.
Patient serum samples
A total of 109 serum samples were collected from Shenzhen Blood Center (Guangdong, China). Each sample was divided into 4 aliquots and frozen to -80°C within 2 h of receiving [36
]. These samples were used to compare the performances of BIOER HCV real-time RT-PCR fluorescence detection kit (Hangzhou BIOER Technology Co. Ltd., Hangzhou, China), Kehua HCV RNA real-time RT-PCR detection kit (Shanghai Kehua Bio-Engineering Co. Ltd., Shanghai, China), qualitative duplex real-time RT-PCR assay, and COBAS AmpliPrep (CAP)/COBAS TaqMan (CTM) assay (Roche Molecular Systems, Pleasanton, CA).
A total of 100 HCV-negative serum samples were obtained from blood donors, including those with hepatitis A, hepatitis B, hepatitis E, human immunodeficiency virus type 1 infection, and human T-cell leukaemia virus infection (confirmed at the blood bank), and negative serum samples obtained from normal persons were used for determining the specificity of the duplex real-time RT-PCR assay.
Further, 40 HCV serum samples were collected from Beijing Blood Center (Beijing, China); the samples included 30 HCV-positive and 10 HCV-negative samples (confirmed at the centre). These samples were used for comparing the performances of singleplex primer/probe and duplex primer/probe assays.
HCV sequences were aligned using sequence comparison software. Based on the consensus sequences of the HCV genome, 2 sets of primers/probes were designed, which, in combination, could detect all the HCV sequences recorded in the Los Alamos National Laboratory HCV Sequence Database [37
]. Probes for the detection of HCV and IC were labelled with 6-carboxyfluorescein (FAM) and a cyanine dye, Cy5, at the 5' end, respectively (Table ).
Primers/probes used in the study
Construction of IC
IC sequences were identical to the wild-type HCV sequences, except for the probe Ap- and probe Bp-binding site sequences, which were replaced by the internal probe sequences (Figure ). Gene splicing by overlap extension PCR was performed to construct an IC sequence containing 3 fragments (Figure ). The overlap extension PCR product was cloned into the plasmid pACYC-MS2 [38
] (constructed at our laboratory) and then verified by sequencing. The plasmids pACYC-MS2-IC were transformed into competent Escherichia coli
BL21 (DE3) strains. After expression and purification, the armored RNA was harvested and quantified.
Figure 4 Construction of IC by using overlap extension PCR. (a) The internal probe-binding sequences were introduced into the HCV 5' UTR sequence by 3 cycles of PCR using primers designed by amplifying overlapping regions. (b) Constructed IC sequence. The blue (more ...)
In order to determine the optimal concentration of IC used in the duplex real-time RT-PCR assay, the armored RNA was serially diluted and then spiked into the national reference material for HCV RNA (GBW09151; 2.26 × 102 IU/ml, 2.26 × 103 IU/ml, 3.97 × 104 IU/ml, 8.5 × 105 IU/ml). Thereafter, it was coextracted and coamplified with the samples in the same reaction tube.
Nucleic acid extraction
RNA was extracted from 0.1-ml sample by using extraction reagents of the Kehua HCV RNA real-time RT-PCR detection kit (Shanghai Kehua Bio-Engineering Co. Ltd.) according to the manufacturer's instructions. The extracted RNA was eluted in 20 μl of diethyl pyrocarbonate-treated H2O and used as the template for the duplex real-time RT-PCR assay.
Duplex real-time RT-PCR amplification for HCV RNA detection
The duplex real-time RT-PCR assay was performed on the ABI PRISM system (Applied Biosystems, America) by using 10 μl of RNA (using extraction reagents of the Kehua HCV RNA real-time RT-PCR detection kit) in a 25-μl volume containing 12.5 μl of 2× QuantiTect Probe RT-PCR Master Mix and 0.25 μl of QuantiTect RT Mix (QIAGEN, German). In the singleplex mode, either the primer/probe set A or the primer/probe set B was used in the reaction, while in the duplex mode, both the primer/probe sets A and B were used in RT-PCR. Armored RNA particles, added to each sample prior to extraction, were used as ICs in the extraction and amplification processes.
Comparison between singleplex primer/probe and duplex primer/probe real-time RT-PCR assays for HCV RNA detection
The 40 serum samples collected from Beijing Blood Center were tested by singleplex primer/probe and duplex primer/probe assays, and the results were then compared.
Commercial kits for HCV RNA detection
A total of 109 serum samples were tested using BIOER HCV real-time RT-PCR fluorescence detection kit (Hangzhou BIOER Technology Co. Ltd.), Kehua HCV RNA real-time RT-PCR detection kit (Shanghai Kehua Bio-Engineering Co. Ltd.), and CAP/CTM assay kit. All the operation steps were carried out according to the instructions given in the manuals provided by the manufacturers.
(i) Detection using BIOER HCV real-time RT-PCR fluorescence detection kit. HCV RNA was recovered from 900-μl of serum and quantified in the LineGene real time PCR assay system, according to the manufacturer's instructions. The results were determined based on the Ct values. The LOD of BIOER HCV fluorescence detection kit was 500 IU/ml.
(ii) Detection using Kehua HCV RNA real-time RT-PCR detection kit. HCV RNA was extracted from 100-μl sample and eluted in 20-μl of diethyl pyrocarbonate-treated H2O. 12.5-μl extract was used as the template in 25-μl reaction. RT-PCR was carried out in a 32-well Lightcycler thermal cycles system (Roche). The LOD of Kehua HCV RNA assay kit was 500 IU/ml.
(iii) Detection using CAP/CTM HCV assay kit. The CAP/CTM test utilizes automated specimen preparation on the COBAS AmpliPrep Instrument by a generic silica-based capture technique. 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. The Cobas TaqMan 48 Analyzer was used for automated real-time RT-PCR amplification and detection of PCR products, simultaneously. HCV RNA levels were expressed in IU/ml. The LOD of CAP/CTM HCV assay was 15 IU/ml.
Results are expressed as mean and standard deviation (SD), as appropriate. The intra-assay and inter-assay variations are expressed as SD and coefficient of variation (CV), based on the mean Ct values. Probit analysis was performed to determine the LOD. The LOD was determined as 95% probability of obtaining a positive HCV RNA result. Correlation coefficients (R) were calculated for linearity data.