[Development of a method to detect serum unmethylated insulin DNA]

Objective: To develop a specific and sensitive method to detect serum unmethylated insulin (INS) DNA for monitoring pancreatic beta cell death and evaluate its clinical practice value. Methods: This study validated and modified the Akirav method and the Husseiny method. Six type 1 diabetes mellitus (T1DM) patients, who were diagnosed with T1DM in the Second Xiangya Hospital, Central South University between February 2014 and February 2015, and another 8 sex- and age-matched healthy controls, were enrolled in the study. Patients' serum samples were examined by the modified Akirav method. Results: Methylation-independent PCR of the Akirav method cannot amplify the specific product. The efficiency of primers for quantitative methylation-specific PCR used in Husseiny method was (76.98±2.88)%, which was lower than 80% (recommend efficiency), resulting in poor reliability. This study applied primers for the Husseiny method into the procedures of the Akirav method.The results showed that the level of unmethylated INS DNA in T1DM group is significantly higher than that in the healthy controls[demethylation index: 0.039(0.028-0.083) vs 0.016(0.003-0.029), P=0.005]. Conclusions: The modified Akirav method can effectively detect the level of unmethylated INS DNA. The level of unmethylated INS DNA in T1DM patients is higher than that in the healthy controls.