Establishment And Verification Of Fluorescence Quantitative PCR For Detection Of Recombinant Oncolytic Herpes Simplex

Objective:To establish an efficient and sensitive fluorescence quantitative PCR method for the detection of recombinant oncolytic herpes simplex virus type 2?oHSV2?.The method was preliminarily verified according to the guiding principles of methodological verification in Chinese Pharmacopoeia,and detected the expression of GM-CSF in tumor tissues of mice after giving medicine.Methods:?1?By analyzing the whole genome sequence of oHSV2 and using Beacon Designer software,specific primers were designed according to the GM-CSF gene fragment.The positive control plasmid DNA was extracted and diluted to the standard for fluorescence quantitative PCR after the determination of the concentration and the conversion.A standard curve was created.The sensitivity,specificity and reproducibility of the method were tested.?2?The blood and urine of donors were collected and mixed with 10³?10⁴?10^5.5?10^6.5 copies/?L pHG52d34.5CMVhGM-CSF plasmid.The total DNA was picked up with QIAamp DNA Mini kit and SYBR Green real-time PCR reaction was carried out to preliminarily verify the method.?3?The A375 cell line was implanted into subcutaneous tumor in nude mice.After the tumor grew to a certain volume,oHSV2 was given to the tumor.Samples were taken at 24 h?48 h?72 h?96 h respectively,and the RNA of which was extracted.PCR detection was carried out after reverse transcription.Results:The lowest detection limit of oHSV2 was 10³ copies/?L,the standard curve equation was y=-3.283log?x?+40.328,R² was 0.998 and the coefficient of variation within and between batches was 0.357%¹.156%and 1.06%³.35%respectively.The recovery of the pre-added plasmid in blood was 96.6%¹09.3%.The recovery of the pre-added plasmid in urine was 96.9%¹04.7%.The expression of GM-CSF can be detected in 24 h?48 h?72 h tumor samples,and the result of 96h is lower than the lower limit of detection.Conclusion:?1?The fluorescence quantitative PCR method for the detection of oHSV2 was successfully established,which can provide theoretical and technical support for the follow-up clinical trials.?2?The PCR method for detecting oHSV2 was verified.?3?This method can detect the expression of GM-CSF in tumor tissue of mice after giving medicine.