A type-specific nested PCR assay established and applied for investigation of HBV genotype and subgenotype in Chinese patients with chronic HBV infection.
BACKGROUND
Many studies have suggested that hepatitis B virus (HBV) genotypes show not only geographical distribution and race specificity, but also are associated with disease progression and response to interferon treatment. The objective of this study was to develop a nested polymerase chain reaction (nPCR) assay for genotypes A-D and subgenotypes B1, B2, C1 and C2 of hepatitis B virus (HBV) and to investigate the distribution characteristics of HBV genotypes/subgenotype in China.


METHODS
After redesigning the primers and optimizing the reaction conditions using common Taq polymerase, the sensitivity, specificity and reproducibility of the method were evaluated using plasmids and serum samples. In total, 642 serum samples from patients with chronic HBV infection were applied to investigate the distribution of HBV genotype and subgenotype in China.


RESULTS
The genotype and subgenotype could be identified when the HBV DNA load of a sample was ≥10(2.3) IU/mL. For the 639 successfully genotyped samples, the sequencing results of 130 randomly selected samples (20.3%, 130/639) were consistent with those of the nPCR method. The present study showed that HBV genotype B (11.2%, 72/642), C (68.2%, 438/642) and D (7.2%, 46/642) were circulating in China, while genotype C was the dominant strain except for western region where genotype D was the prevalent strain. The main subgenotypes of genotypes B and C were B2 (87.5%, 63/72) and C2 (92.9%, 407/438), respectively.


CONCLUSIONS
The low-cost nPCR method would be a useful tool for clinical and epidemiological investigation in the regions where genotypes A-D are predominant.
Department of Microbiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China.
Jing-Jing
JJ
Nie
Kui-Xia
KX
Sun
Jie
J
Li
Jie
J
Wang
Hui
H
Jin
Ling
L
Wang
Feng-Min
FM
Lu
Tong
T
Li
Ling
L
Yan
Jing-Xian
JX
Yang
Mi-Shu
MS
Sun
Hui
H
Zhuang
D044466:Asian People; D002681:China / Q000453:epidemiology; D017931:DNA Primers; D005838:Genotype; D006515:Hepatitis B virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000453:epidemiology / Q000821:virology*; D006801:Humans; D017720:Molecular Epidemiology; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014773:Virology / Q000191:economics / Q000379:methods*
D017931:DNA Primers