[Establishment and preliminary application of a gene chip for detection of hepatitis B virus "a" determinant hotpoint mutation].
OBJECTIVE
To develop a gene chip for rapid detection of the "a" determinant hotpoint mutation of hepatitis B virus (HBV).


METHODS
Primers were designed in the HBV conservative region, and probes for detecting 126A, 126S, 144A, 145R, 145E, 144A+145R, and 144A+145E mutants were developed for that gene chip. PCR amplification and gene chip technology were optimized. The performance of the gene chip was evaluated by detecting the reference plasmids. Forty five samples of serum obtained from patients with chronic hepatitis B were used to compare the sensitivity of the gene chip and the direct sequencing of PCR products.


RESULTS
The oligonucleotide microarray was specific for mutant and native plasmids. The sensitivity of the gene chip was 5 x 10(3)copies/micro l with a high reproducibility. The gene chip could detect minor variants when they were more than 10% among the HBV strains. The positive rates of 126A, 126S-1, 126S-2 detected in the 45 specimens by the gene chip (46.67%, 35.56% and 24.44%, respectively) were higher than those detected by direct sequencing of PCR products (9.00%, 4.44% and 2.22%; P=0.000, P=0.000 and P=0.002, respectively). The sequencing of cloned PCR products demonstrated that the gene chip was specific for the "a" determinant hotpoint mutation detection.


CONCLUSIONS
HBV "a" determinant hotpoint mutations can be detected by oligonucleotide microarray with high sensitivity and specificity, providing a method for large scale screening of the mutants.
Zhang
Rui
R
National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China.
Li
Rong-cheng
RC
Li
Yan-ping
YP
Wang
Sheng-qi
SQ
Liang
Zheng-lun
ZL
Li
He-min
HM
Zhuang
Hui
H
D006509:Hepatitis B / Q000097:blood / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017354:Point Mutation