Establishment Of The First National Standard For Nucleic Acid Amplification Technology (NAT) Assay For HCV RNA

Background and Objective To establish an international standard for nucleic acid amplification technology(NAT) assay for HCV RNA in China. To develop an expression system producing the virus-like particles (VLPs) which contain the HCV recombinant RNA (reRNA). The reRNA within VLPs is ribonuclease resistant due to the encapsidation by MS2 bacteriophage coat proteins.Methods Real-time PCR tests were performed to screen the positive HCV plasma, and then the positive samples were selected and diluted with negative plasma in which fibrinogen has been eradicated. The sample were lyophilised with a concentration of approximately 300,000 copies/ml. The assay method used included the Roche Amplicor assay (version 2.0) and real-time PCR. The lyophilised preparation were calibrated by the international standard (NIBSC code:96/790) from NIBSC. According to the 5' UTR sequence of HCV genotypes 1,2 and 6, which were the prevalent genotypes in China, we design 2 pair primers to amplify HCV 5'UTR seqences by RT-PCR. A Hindlll restriction sequence was incorporated into the primers.The PCR product was purified and then cloned into T vector. The digested fragments were purified from Gel after digesting the T vector with Hind III restriction enzyme. The pNCCLl previously digested with HindIIIrestriction enzyme was combined with the digested fragment to creat a new expression plasmid pNCCL1-HCV.Under the induction of IPTG, MS2 coat proteins were expressed into the cytoplasma of E.coli strain BL21-DE3 harboring pNCCL1-HCV, and then assembled with HCV reRNA into VLPs. The induced cells were sonicated and then the VLPs were harvested in the sonicate supernatant after centrifugation. In order to examine whether HCV reRNA was packaged into the VLPs, 20ul of sonicate supernatant was diluted from 1:10 to l:10⁶ fold and then RT-PCR was performed after RNA were extracted from dilutions according to the protocol "Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate -Chloroform Extraction". To test the durability of HCV reRNA with the VLPs, 20ul of supernatant was incubated with RNaseA( 100U) and DNaseI(20U)at 37℃ for 4d .20μl of sonicate supernatant was pelleted with PEG600 and resuspended in 20μl of normal human negative serum, then incubated at 45 ℃ for 3d to examine the shipping compatibility.Results The titer of this lyophilised preparation is (1.29±0.24)×l0⁵ IU/ml compare with the International Standard for HCV RNA 96/790. The stability test indicate that the sample were stable at room temperature(20-25℃) for 2 weeks and at 37℃ for at least 1 week. Long-term stability was abserved at 2-8℃ for 6 months and at -20℃ for more tan 2yearswith no significant decrease. The vail-to-vail imprecision is 3.53%. In 2005, the certificates of National Reference materials were issued for the two candidate materials by General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, namely GBW (E) 090032 for HCV RNA. The plasmid packaging system for VLPs containing HCV RNA was successfully constructed, first VLPs containing HCV-1a characteristic parts reRNA,second VLPs containing HCV-2b characteristic parts reRNA, and the third one containing HCV-6a characteristic parts reRNA. The concentration of reRNA within the VLPs is more than 10¹⁰ copies/ml. HCV reRNA had RNase-resistant characters because of the encapsidation by MS2 coat protein. The VLPs in supernatant were durable when incubated at 37℃ for 4d with the digestion of RNase A (100U) and DNase 1 (20U) ,and the VLPs in normal human negative serum incubated at 45 ℃ for 3d were still stable.Conclusions The certificated reference materials was the first national reference materials for nucleic acid test of pathogens in China. Based on the results of this study, the lyophilized sample was established as the first international standard for nucleic acid amplification technology (NAT) assay for HCV RNA in China. Above all, the two reference materials have been widely used by related major domestic reagent manufactures and the stability and accuracy have been confirmed. The calibrators in commercial kits which are traceable to these two reference materials can eradicate the difference caused by different kits or different staffs and then make the results comparable. Therefore it is very promising for the reference material to be applied in HCV RNA quantitative tests, and then facilitate the standardization of the HCV RNA quantitative tests. The expression plasmid vector pET-MS2-HCV can be used as a platform to produce noninfectious and RNase-resistant HCV-1b, HCV-2a and HCV-6a RNA quantitative standards and controls. The VLPs can be directly used as external standards for HCV RNA quantitative RT-PCR kits and can also act as positive controls for HCV RNA qualitative tests. Moreover, the VLPs can be used to set up samples panel for evaluation of HCV RT-PCR kits and can act as the non-infectious proficiency standards for the laboratories involved in HCV RNA test. Additionally, after some modifications of target cDNA such as mutation, deletion and insertion, the VLPs containing modified reRNA can be used as internal quantitative standards and internal controls of the RT-PCR.