[Establishment and application of a real-time PCR method to detect hepatitis B virus cccDNA quantitatively].
OBJECTIVE
To establish a new method to detect HBV cccDNA quantitatively and to apply it to detect cccDNA in liver needle biopsy specimens of chronic hepatitis B patients.


METHODS
The sequences of HBV DNA genotypes A through G were analyzed. According to the different sequence structure of cccDNA and rcDNA, primes and probe were designed in highly conservative region outside the nick of cccDNA in order to amplify cccDNA but not rcDNA. The best conditions of this method were found after testing experiments. Also we checked its specificity and sensitivity and reproducibility. The products of PCR were sequenced in order to ascertain if it was the right region expected. To amplify with standard plasmid ranged from 10(2) to 10(10) copies/ml to measure the sensitivity and amplify in parallel with standard plasmid of 10(6) copies/ml for 30 replicates so as to measure its reproducibility. DNA was extracted from 32 needle liver biopsy specimens of chronic hepatitis B patients. The cccDNA was quantitatively detected with this method. The data of cccDNA obtained before and after therapy and their relationship with total HBV DNA were analyzed. RESULTS Results of sequencing showed that the PCR product was from the right region. The sensitivity was 10(3)-10(10) copies/ml. The Ct value was 29.69+/-0.31 and the coefficient of variability was 1.04 percent calculated from the data of 30 PCR reactions with standard plasmid. The percentage of decrease in serum HBV DNA, total HBV DNA in liver and cccDNA in liver were 0.49+/-0.17, 0.22+/-0.18 and 0.16+/-0.28 respectively. There is 47 percent-98 percent cccDNA in total HBV DNA in liver and the mean is 81.5 percent.


CONCLUSIONS
The method is good because of the simple and convenient operation, the high specificity, the wide linear detection range and the fine reproducibility. Therefore it can be used for both scientific research and clinical purpose. Lamividine can significantly inhibit serum HBV DNA by, but its inhibitory effect on cccDNA in liver was rather weak.
Zhuang
Li-wei
LW
Department of Infectious Diseases, The First Hospital, Peking University, Beijing 100034, China.
Lu
Hai-ying
HY
Yu
Yan-yan
YY
Si
Chong-wen
CW
Yu
Min
M
Zhang
Nai-lin
NL
Gong
Wei-bo
WB
D004270:DNA, Circular / Q000235:genetics; D004279:DNA, Viral / Q000097:blood / Q000235:genetics*; D006509:Hepatitis B / Q000175:diagnosis / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004270:DNA, Circular; D004279:DNA, Viral