Standad Materials Based On Armored RNA For BCR-ABL Fusion Gene:Establishment And Application

BCR-ABL fusion gene is the molecular pathogenesis mechanism of chronic myelogenous leukemia and the basis for diagnosis and targeted therapy.To improve the detection level of BCR-ABL transcripts,International standards(IS)were established for detection and quantification of BCR-ABL transcripts by the World Health Organization(WHO).For reagent manufacturers,the IS can be used to determine the Performance verification of their test reagents and calibrate the assays;For clinical laboratories,the IS can be used to Analysis of sensitivity of the reagent and internal quality control;For researchers,the IS can be used to make domestic reference materials which can be traced to international standards.However,the International standards were expensive,the resources were limited,so it’s not enough for calibration and evaluate the testing capabilities of clinical laboratories.In addition,the external quality assessment is important for BCR-ABL gene detection.The external quality assessment of the fusion gene is still blank Before our study,and effective assessment were lacked for the standardization of reagents,external quality assessment and the accuracy of clinical laboratories.The cDNA fragment of maturation protein,coat protein,packaging cites of MS2,the fusion gene of P210 BCR-ABL and the reference gene of ABL was cloned into vector pACYCDuet-1.Then the Amored RNA,encapsidated with p210 and control gene respectively in the presence of bacteriophage coat proteins,was purified by cellufine sulfate column and subsquently identified by RT-PCR and RT-qPCR.The VLPs containing target sequences from p210 BCR-ABL and control were constructed.Then the concentration of p210-VLP and CG-VLPs were initially valued,and individually packed in vials were done after the VLPs were mixed.Then the ratio of p210/CG transcripts level were valued according to standard cured established with ARQ IS calibrators after safety and stability detection.The results show that the specific sequences of exogenous fragments can be contained into MS2 by Armored RNA.It is resistance to RNase enzyme degradation,the laboratory biosafety requirements of Armored RNA are low,and therenis a highly stable and homogenously characteristics.They could be reproducibly used for up to 8 months when stored at-20℃,up to 3 month stored at 4℃,up to 15 days stored at room temperature,and up to 7days stored at 37°℃.The concentration ratio were 23.8%,2.64%,0.27%,0.027% respectively when the control gene was ABL;the concentration ratio were 22.0%,2.29%,0.20%,0.021% respectively when the control gene was BCR;the concentration ratio were 19.0%,2.02%,0.22%,0.020% respectively when the control gene was GUS.These results suggest that the traditional MS2 phage cloning program to encapsulate longer foreign target RNA sequences into MS2 phage envelope proteins can be successfully used to constructing novel standards for fusion gene detection.Clinical and laboratory applications were expanded while three control genes were contained.Also the development of RNA nucleic acid standards were further promoted.Armored RNA served as standard material for monitoring the test of BCR-ABL transcript level were an ideal reference material and has potential clinical applicability.