Application of Sanger sequencing technology in detection of gemcitabine resistance sensitive gene CDA / 国际检验医学杂志

ABSTRACT

Objective To analyze the polymorphic site mutation of gemcitabine resistant sensitive gene cytidine deaminase (CDA),and to investigate the applicability of the Sanger detection technology in clinical sample detection with the real-time fluores-cence quantitative PCR as the reference.Methods The peripheral blood samples from 255 cases of stage ⅢB to Ⅳ non small-cell lung cancer(NSCLC)were randomly selected for extracting DNA.The G208A and A79C mutation situation of CDA gene was re-spectively detected by qPCR and Sanger sequencing technology.Results The success rate for detecting the CDA gene mutation in peripheral blood sample in 255 cases of stage ⅢB and Ⅳ NSCLC were 98.04%(250/255)by the Sanger sequencing method and 97.25%(248/255)by qPCR.With the qPCR as the reference,the sensitivity and specificity of Sanger sequencing method was 92.50% and 99.52% respectively.All mutation types detected by two methods were fully consistent.Conclusion Adopting the Sanger sequencing method can effectively detect drug resistance gene CDA mutation,realizes the mutation typing,can accurately, high throughput and efficiently detect CDA mutation site,which provides an effective detection method for gemcitabine sensitive medication of NSCLC.