Establishment of qPCR method for detection of oncolytic virus product GC001 and its biodistribution in mice
Objective To establish a universal qPCR analysis method to investigate the biodistribution of oncolytic virus product GC001 in tissues and organs,and to detect samples collected during the concomitant biodistribution study of the repeated-dose toxicity in BALB/c mice.Methods Primers and probe were designed according to the specific sequence of drug,the impacts originated from incorporated genome of different tissues and/or animal species on PCR reaction system were investigated,and the standard curve for detection was constructed and verified methodologically.Ninety BALB/c mice were randomly divided into three groups consisting of control group(normal saline),and GC001 low-and high-dose groups(9.7 × 106,1.51 × 108 pfu),with fifteen animals per sex per group.The animals were subcutaneously injected once every two weeks for a total of three doses followed with an eight-week recovery phase.On day 18,day 31(2 d after the end of dosing)and day 85(the end of recovery period),tissues and organs were collected including blood,gonad,kidney,liver,lung,heart,brain,spleen,lymph nodes(mesenteric and inguinal),injection site,skin,bone marrow,spinal cord and sciatic nerve and subjected to qPCR to determine the copy number of the drug.Results A universal standard curve named pEASY was constructed with the limit of quantitation(LOQ)of 10 copies/µL and quantitative range between 1.0 × 101 and 1.0 × 107 copies/μL,which manifested excellent precision,repeatability and consistency.GC001 spread broadly in the body of mice and achieved many tissues and organs,and mainly accumulated in the injection site,inguinal lymph node,sciatic nerve,spinal cord,skin,bone marrow and blood,which declined gradually with time extension and wouldn't persist perpetually.Its systematical distribution was thought of the reason of blood flow.Conclusion This study revealed the distribution profile and metabolic trends of WR strain vaccinia virus product GC001 in mice,which has certain reference significance for the biological distribution research of other vaccinia virus carrier drugs.
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