| ObjectiveTo establish a duplex real-time polymerase chain reaction (PCR) assay avoiding missing detection as many as possible. At same time, we need construct an internal control (IC) resistant to DNase and noninfectious to humans, avoiding false negative results.MethodsAll the sequences of HBV recorded in the Los Alamos National Laboratory HBV Sequence Database were aligned, and the primers and probes were designed in the conserved region of HBV, then the real time PCR assay for the detection of HBV DNA was taken shape initially. According to the basic principle of probe design, we designed 6 sets of primers/probes in the conserved region of HBV. By means of detection of clinical samples, we screened out 2 sets of primers/probes with the best performance in the HBV DNA assay.The IC was constructed by use of the overlapping extension PCR technique. IC sequences were identical to the wild-type HBV sequences, except for the probe binding site sequences, which were replaced by the internal probe sequences. We used traditional lambda phage cloning procedure to construct the stable armored DNA that can function as the IC for HBV DNA assay. The obtained armored DNA was serially diluted and spiked into the national reference material for HBV DNA assay, then we determined the optimal concentration of IC used in the duplex real-time RT-PCR assay.A dilution series of the World Health Organization (WHO) International Standard for HBV DNA (National Institute for Biological Standards and Control (NIBSC), code 97/750, UK) was used to determine the limit of detection (LOD) of the duplex real-time PCR assay. Linearity of the duplex real-time 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. numerous clinical samples were used to compare the performances of single primer/probe assay, Kehua HBV DNA real-time PCR detection kit (Shanghai KeHua Bio-Engineering Co. Ltd., Shanghai, China) and qualitative duplex real-time PCR assay.ResultsWe successfully established the duplex real-time PCR assay with armoured DNA as internal control. The limit of detection of the duplex primer/probe assay was 29.5 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. In the testing of 69 serum samples, the performance of the duplex real-time PCR assay was identical to that of the COB AS AmpliPrep (CAP)/COBAS TaqMan (CTM) HBV assay and superior to the commercial HBV assay kits. We constructed an armored DNA particle that can function as the internal control. During construction, we used the lambda phage gt11 vectors and EcoRI restriction enzyme cutting sites in the traditional lambda phage cloning procedure. The obtained armored DNA was noninfectious to humans, resistant to DNase I digestion and exhibited improved storage and handling properties. In the study,1000 copies/ml of armored DNA was used as the optimal concentration in the duplex real-time PCR assay.ConclusionThe lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard.The duplex real-time PCR assay is an efficient and effective viral assay. It is appropriate for blood-donor screening and laboratory diagnosis of HBV infection. Furthermore, the cost of the duplex real-time PCR assay was considerably lower than that CAP/CTM HBV assay kits, and hence, the former assay is more suitable for large-scale use. ObjectiveTo establish a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay avoiding missing detection as many as possible. At same time, we need construct a specific armored RNA as internal control (IC), avoiding false negative results.MethodsAll the 5'UTR sequences of HCV recorded in the Los Alamos National Laboratory HCV Sequence Database were aligned, and the primers and probes were designed in the conserved region of HCV, then the real time RT-PCR assay for the detection of HCV RNA was taken shape initially.According to the basic principle of probe design, we designed 10 sets of primers/probes in the 5'UTR sequences of HCV. By means of detection of clinical samples, we screened out 2 sets of primers/probes with the best performance in the HCV RNA assay.The IC was constructed by use of the overlapping extension PCR technique. IC sequences were identical to the wild-type HCV sequences, except for the probe binding site sequences, which were replaced by the internal probe sequences. Armored RNA was successfully used as IC in the duplex real-time RT-PCR assay. In order to avoid the suppression of target amplification, the concentration of armored RNA spiked into the samples was optimized.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. 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. 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. A total of 109 serum 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).ResultsThe limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP)/COBAS TaqMan (CTM) assay and superior to 2 commercial HCV assay kits.ConclusionThe duplex real-time RT-PCR assay is an efficient and effective viral assay. It 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.
|
PDF Full Text Request