Establishment and evaluation of a TaqMan RT-qPCR method for tissue quantification of oncolytic virus M1 in vivo

AIMTo establish a TaqMan RT-qPCR method for surveiling the spread of oncolytic virus M1 in tissue, helping control the dosage and assessing the safety of virus. METHODSA TaqMan-based one-step RT-qP?CR method for the detection and quantification of oncolytic virus M1 in the tissues was established. The virus load and distribution in the tissues of SD rats, cynomolgus monkeys and nude mice were also investigated. RESULTSA pair of specif?ic primers (Q3) and the standard viral RNA for SYBR Green RT-qPCR were screened and selected with the best specificity and amplification efficiency. By optimizing the experiment conditions, we found that the annealing temperature above 62℃ reduced matrix effect but affected the amplification efficiency. So we established a one-step TaqMan RT-qPCR method and redesigned a pair of Q3 short primers (Q3S). Using the one-step TaqMan RT-qPCR and Q3S primer, the standard RNA with low copy numbers was specifically detected under the background of mixed matrix RNA of SD rats or cynomolgus monkeys. Furthermore, the method was verified to be suitable for detecting tissue distribution of M1 virus in the mice, SD rats and cynomolgus monkeys. CONCLUSION The TaqMan-based one-step RT-qPCR constructed with Q3S primer can be used for M1 virus quantification in various tissue samples of different animals with better specificity and sensitivity, and may be further applied to the detection of clinical samples.