Abstract
A SYBR Green I real-time quantitative RT-PCR method was established for investigating the correlation between CML28 mRNA expressing levels and relapse of leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitorting of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph+ ALL. The sensitivity of the established method was at 10−4 level, with interassay variation and intraassay variation of standard samples both < 10%. The CML28 was highly expressed in AML and CML-BP or AP. In newly diagnosed group, CML28 was (6.58±2.34)×10−2. In pre-conditioning regimen group was (2.19±0.32)×10−2, in group that 1 month after allo-HSCT was (1.35±1.28)×10−2, in group that 3 months after allo-HSCT was (4.57±6.39)×10−3. CML28 can be detected 3months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2×10−2) survived without relapse, the other 2 patients with high level (>2×10−2) relapsed within one year,1 died and1 received the second time allo-HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2×10−2 and relapsed again. CML28 mRNA level was obviously correlated with the development of diseases. Serial quantification of CML28 mRNA levels were necessary for allo-HSCT recipients, and more informative than a single detection. Use of this assay to evaluate MRD in the patients performed allo-HSCT was helpful for predicition of relapse.
Development of a novel real-time RT-PCR assay with LUX primer for the detection of swine transmissible gastroenteritis virus