Pyrosequencing of pre-C/BCP mutation in hepatitis B
- VernacularTitle:焦磷酸测序法检测HBV前C区/基本核心启动子突变的临床应用
- Author:
Lan YAO
;
Jiawu SONG
;
Zhi XIN
;
Xiaofeng LI
;
Zhouqing WU
;
Jianxi TANG
;
Bo GUO
;
Bo WU
;
Renxu LAI
- Publication Type:Journal Article
- Keywords:
Hepatitis B virus;
Promoter regions ( genetics );
Genes,viral;
Mutation;
Phosphoric acids;
Polymerase chain reaction;
Oligonucleotide array sequence analysis
- From:
Chinese Journal of Laboratory Medicine
2008;31(8):860-863
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a clinically useful assay for detecting the mutations of HBV pre-C/BCP based on the pyrosequencing and accuracy, reproducibility and reliability of this assay was evaluated. Methods The pyrosequencing primers for HBV pre-C/BCP mutation were designed through the cluster analysis among one hundred HBV gene sequences. After the amplification of the fragment of pre-C/BCP with the template of pre-C/BCP mutation plasmids, the pyrosequencing method for pre-C/BCP detection was initially set up with this standard sample. The accuracy, reliability and reproducibility of the pre-C/BCP pyrosequencing were confirmed through the pre-C/BCP plasmids as a standard sample when compared with Sanger/genechip sequencing method pre-C/BCP pyrosequencing assay was applied for detecting pre-C/BCP mutation types of 60 chnical serum samples in HBV patients. Results The pre-C/BCP mutation detection assay based on pyrosequencing has been established in our study. The coincidence rate between pyrosequencing and Sanger squencing was 100%. The coincidence rate between the result of pyrosequencing and of genechip method was 91.7%. The reproducibility of this assay was 97. 8%. It indicates the pre-C/BCP pyrosequencing is a high-accurate method with, good-reproducibility and high-reliability. And multi-site detection can be achieved by pyrosequencing one time. A rare mutation T1758C was also detected. Conclusion Pyrosequencing for pre-C/BCP mutations assay is high-throughout method for simultaneous detection of multi-site mutation.
