RESUMO
Objective To develop a TaqMan fluorogenic probe based amplification refractory mutation system (TaqMan-ARMS) for the detection of the C BS gene G919A and T833C mutations,and to investigate whether the mutations are associated with diabetic nephropathy in Chinese population. Methods According to the principle of amplification refractory mutation system,the cycle threshold (Ct) of wild (Wct) and mutation (Mct) allele-specific primers between the two PCR reactions in real-time PCR were monitored and genotype detection criteria were established based on the threshold ratio Act (?ct= Wct/ Mct) or an appearance of exponential amplification.With this technique,the G919A and T833C mutations in the CBS gene were analyzed in 94 patients with diabetic nephropathy and 140 control subjects. Results The detection criteria of TaqMan-ARMS assay for T833 allele were ?ct40,for T833C allele 0.940,respectively.The criteria for G919 allele were ?ct40,for G919A allele 0.9240,respectively.The T833C rates of TT,TC and CC genotypes were 98.94%, 1.06% and 0 in the patients and 99.29%,0.71% and 0 in the controls.The 833C allele frequencies were 0.53% in the patients and 0.36% in the controls.No significant differences in both genotypes and allele of T833C mutation were observed between the two groups.The G919A point mutation was not observed in all subjects.Conclusions A TaqMan-ARMS assay for the detection of the CBS gene GA919A and T833C mutations has been developed.The assay is accurate and sensitive,and is suitable for high-throughput detection of the point mutations.The G919A and T833C point mutations of CBS gene may not be related to diabetic nephropathy in Chinese.