Detection of XPO1(E571K) Gene Mutation from Cell-Free DNA in Blood Circulation of Lymphoma Patients by FAST-COLD PCR.

The XPO1 (exportin 1) gene encodes exportin 1 protein responsible for transporting proteins and RNA from the nucleus to the cytoplasm. It has been used as a biomarker for lymphoma detection. XPO1(E571K) mutation has been frequently observed and identified as a good prognostic indicator for lymphoma patients. The detection of a target molecule released by lymphoma cells into blood circulation (cell-free circulating tumor DNA, cfDNA) is a better method than tissue biopsy. However, cfDNA concentration in blood circulation is very low in cancer patients. Therefore, a precise and sensitive method is needed. In this study, cfDNA was extracted, and then the XPO1 gene was detected and amplified using conventional PCR. Sanger sequencing was employed to verify the DNA sequences. FAST-COLD-PCR was developed to detect XPO1(E571K) gene mutation using a CFX96 Touch Real-Time PCR System. The optimal critical temperature (Tc) was 73.3 °C, allowing selective amplification of XPO1(E571K) mutant DNA while wild-type XPO1 could not be amplified. XPO1(E571K) gene mutation can be detected by this method with high specificity and sensitivity in lymphoma patients. This approach facilitates rapid and straightforward detection in a timely manner after the diagnosis. Accordingly, the optimized FAST-COLD-PCR conditions can be used as a prototype for XPO1(E571K) mutant detection in lymphoma patients.