The Construction Of Virus-like Particles Containing HCV Genotype 1b, 2a And 6 RNA Sequences And The Research On Determination Of HCV Three Mainly Genotypes In China By Real-time Reverse Transcriptase-PCR

ObjectiveTo develop the Non-infectious and RNase-resistant 1b, 2a and 6 HCV virus-like particles (VLPs), which were used for positive controls, external quality assessment (EQA) and genotype detections for clinical labs. Meanwhile, we established a new real-time PCR assay for determination of HCV genotypes, and used the VLPs as an internal control.Methods1. The VLPs containing HCV genotype 1b, 2a and 6 RNA sequences.According to the 5' UTR and NS5b sequences of HCV genotypes 1b,2a and 6,which are the prevalent genotypes in China, we designed primers to amplify HCV 5'UTR and NS5b regions by RT-PCR. Restricted sequences were incorporated into the primers. The PCR product was purified and then cloned into T-easy vector. The digested fragments were purified from Gel after digesting the T-easy vector with restriction enzyme. The pET-MS2 previously digested with restriction enzyme was combined with the digested fragment to create a new expression plasmid. Under the induction of IPTG, it assembled with HCV reRNA into VLPs. Then, we examine whether HCV reRNA was packaged into the VLPs.2. The study in the stability of VLPs and the EQA for HCV RNA test in clinical labsThe VLPs with different concentration were quantified three times with commercialkits for PCR detection. Meanwhile, the stability data of VLPs in different conditions were summarized and evaluated. The EQA involved in HCV RNA test was developed as well. Serum panels were delivered to the clinical laboratories which performed HCV RNA detection in China. Each panel made up of 5 coded samples. All laboratories were requested to carry out the detection within the required time period and report on testing results. All the positive samples were calibrated against the 1st National Standard for HCV RNA.3. The research on determination of HCV three mainly genotypes in China by real-time reverse transcriptase-PCRWe searched HCV 1b, 2a and 6 sequences from NCBI website. After the alignment of each genotype sequences of HCV by BioEdit software, we determined the consensus sequences of HCV genome. With regard to the consensus sequences, oligonucleotides were designed with Primer Express 3.0 software. Then the real-time reverse transcriptase-PCR for HCV genotyping was primary developed.Results1. The construction of virus-like particles containing HCV genotype 1b, 2a and 6 RNA sequences.The recombinant plasmids for 3 genotypes VLPs containing HCV RNA 5'UTR and NS5b regions were successfully constructed. The experiment has proved that the VLPs are surely packaging RNA.2. The study in the stability of VLPs and the EQA for HCV RNA test in clinical labs.The VLPs concentration were quantified against the first National Standard. The stability testing data indicated the quality control materials were stable at least for one month when stored at4℃,-20℃, room temperate, 37℃, -70℃and -70℃freeze-thaw cycles been repeated 3 times. Frome the result of EQA, we can conclude that the whole results are close to the target value, but different genotypes have different percent of pass. Meanwhile, we compared different reagents and different instruments.3. The research on determination of HCV three mainly genotypes in China by real-time reverse transcriptase-PCRThe specific probes and primers of HCV 3 genotypes were designed according to the alignment of HCV sequences.The VLPs which we have constructed could be tested by the specific probes and primers.ConclusionsThe internal control of HCV 1b, 2a and 6 genotypes which is Non-infectious and RNase-resistance performed good stability, and it would be help to the quality control, EQA and genotyping detection related to HCV RNA test.We primary established HCV genotyping assay, and it could be widely used for the clinical diagnose and therapy in the future.