Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.
OBJECTIVE
To establish a modified microarray method for detecting HBV gene mutations in the clinic.


METHODS
Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry.


RESULTS
HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility.


CONCLUSIONS
An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.
Mao
Hailei
H
Intensive Care Unit, Affiliated Hospital of Nantong University, China.
Wang
Huimin
H
Zhang
Donglei
D
Mao
Hongju
H
Zhao
Jianlong
J
Shi
Jian
J
Cui
Zhichu
Z
D003124:Colorimetry; D004586:Electrophoresis; D006509:Hepatitis B / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D007211:Indoles; D009154:Mutation; D009580:Nitroblue Tetrazolium; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
D007211:Indoles; D009580:Nitroblue Tetrazolium; C035455:5-bromo-4-chloro-3-indoxyl phosphate