General Detection Methods For Five Kinds Of RNA Viruses In Cell Preparations

Cell preparation is an important preparation in cell therapy,which is developing rapidly.Both production management and quality control have strict requirements about cell preparations.RNA viruses in cell preparations,including HCV,HIV-1/2,and HTLV-1/2,are clearly required to be detected,which directly affects the safety and effectiveness of cell therapy,and is also in line with the international mainstream of standardized preparation and application of cell preparations trend.Therefore,this paper studies the general detection methods for these five kinds of RNA viruses in cell preparations.The main contents and results are as follows:1.Virus inactivation effect and viral nucleic acid stability evaluation for cell preparation quality inspection sample preservation solutionThe lentivirus carrying the GFP green fluorescent protein gene was used as the RNA virus model.The RNA virus preservation solutions(A and B)were used for inactivation treatment respectively,and then 293T cells were then infected and cultured for 5 days.The inactivation effect was evaluated by observing the expression of fluorescent proteins in cells,and the nucleic acid stability after virus inactivation was further evaluated by real-time fluorescence quantitative RT-PCR.The results showed that preservation solution B was the best in terms of RNA virus inactivation effect;in terms of RNA virus nucleic acid stability protection,the protective effect of B preservation solution within 6 hours of treating the sample virus was not significantly different from that of the 0h group.This method is helpful for the quality control of RNA virus preservation solution.Comparing the inactivation effect and viral nucleic acid protection effect of different cell preparation quality inspection sample preservation solutions is of great significance for establishing a stable general method for RNA virus nucleic acid detection in cell preparations.2.Preparation and characterization of positive quality controls for RNA virus nucleic acid detection in cell preparationsA positive quality control substance for the detection of HCV,HIV-1/2,and HTLV-1/2 RNA viruses nucleic acids in cell preparations was prepared,that is,virus-like particles(pseudovirus 5V)loaded with the above-mentioned five viral nucleic acids.The conserved gene sequences were determined by downloading genome sequences from international public databases for alignment analysis after epidemiological analysis.The tandem clone for MS2 phage and the above-mentioned viral conserved gene was inserted into the prokaryotic expression vector to construct the p ACYCDuet-1-MS2/5Virus recombinant plasmid,which was then transformed,induced to express and identified;the expression product was purified and concentrated,and the morphology of pseudovirus 5V was analyzed by electron microscopy;the pseudovirus was diluted 5V for DNase I,RNase A resistance and stability tests.The results showed that the recombinant gene was effectively expressed,and the pseudovirus 5V had high purity and no stray bands;the virus gene sequence successfully packaged and designed by the pseudovirus 5V had obvious viral structure characteristics;the pseudovirus 5V could resist the degradation of high concentrations of DNase I and RNase A.Stable for 6 months at-20°C.It shows that the prepared pseudovirus 5V is stable and reliable,and meets the basic requirements as a positive quality control substance for RNA virus nucleic acid detection in cell preparations.3.Preliminary study on real-time quantitative RT-PCR detection methods for five kinds of RNA viruses in cell preparationsViral RNA nucleic acids were extracted from mock samples of cell preparations prepared for pseudovirus 5V.Bioinformatics software was used to design specific primers to establish a real-time quantitative RT-PCR detection method,and the specificity,sensitivity and repeatability of the detection were evaluated respectively.The results showed that the specific primers for HCV,HIV-1/2 and HTLV-1/2 had a good linear relationship between the CT value and the concentration of the corresponding template fluorescence quantitative RT-PCR.Each melting curve showed a single peak,indicating that the primers specifically amplified each segment and the prepared pseudovirus 5V particles had good specificity and high purity.The original copy number of RNA stock solution of pseudovirus 5V is about2.7×10~8copies/μL.The detection limit of real-time fluorescence quantitative PCR for primers specific to different conserved regions was in the range of 10-100 copies/μL.Real-time fluorescence quantitative RT-PCR repeat detection also showed that the standard deviation of CT values amplified by the RNA samples of pseudovirus 5V using each conserved segment as a template were all within 0.5,and the coefficient of variation was below 2%.The amplification curves were bundled,indicating that the five viral nucleic acid templates all had good stability.The results show that the real-time fluorescent RT-PCR method established in this study for the detection of five RNA viruses,HCV,HIV-1/2 and HTLV-1/2,has the potential to be used for the screening and detection for five kinds of RNA viruses in cell preparations.Interpretation of test results still has certain limitations without comparative evaluation based on actual virus-infected cell preparation samples.Conclusively,a general detection method for five kinds of RNA viruses(HCV,HIV-1/2,HTLV-1/2)in cell preparations was carried out in this study.1)Establish a method to evaluate the virus inactivation effect and viral nucleic acid stability of cell preparation sample preservation solution,which is helpful to optimize the sample preservation solution.This method helps to select a preservation solution that can not only protect the nucleic acid quality of viral RNA in the sample but also avoid the sample the RNA virus poses a risk of infection to testing personnel.2)A pseudovirus5V containing conserved regions of the nucleic acid sequences of HCV,HIV-1/2,and HTLV-1/2 RNA viruses was prepared by using armored RNA technology.It can be used as a positive quality control substance in cell preparations.3)Using the cell preparation which containing pseudovirus 5V as a simulated sample,a real-time quantitative RT-PCR detection method for these five viruses was initially established to provide reference for future detection research based on actual virus-infected cell preparation samples.