Abstract
A rapid and sensitive method has been developed to detect hepatitis B virus DNA (HBV) by nested polymerase chain reaction (PCR) technique with primers specific for the surface and core regions in capillary thermal cycler within 80 min. The lower limit for detection by present PCR method is 10−5 pg of recombinant HBV DNA which is equivalent to that determined by one round of PCR amplification and Southern blot hybridization analysis. When boiled HBV positive serum was serially diluted 10-fold, HBV DNA was successfully determined in 1 μl≈10−3 μl of serum. HBV DNA was detected by present method in 69 clinical samples including HBsAg positives and negatives by enzyme-linked immunosorbent assay (ELISA). When serum samples were amplified by nested PCR using surface and core region primers, HBV DNAs were detected in 37 of 69 samples (53.6%) and 18 of 69 samples (26.1%), respectively. These results can inform the infectious state of HBsAg positive pateints. A simple and rapid nested PCR protocol by using boiled serum as DNA template has been described for the clinical utility to determine HBV DNA in human serum.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References Cited
Burckhardt, J., Amplification of DNA from whole blood.PCR Methods Appl., 3, 239–243 (1994).
Chemin, I., Baginski, I., Petit, M. A., Zoulim, F., Pichoud, C., Capel, F., Hantz, O. and Trepo, C., Correlation between HBV DNA detection by polymerase chain reaction and pre-S1 antigenemia in symptomatic and asymptomatic hepatitis B virus infections.J. Med. Virol., 33, 51–57 (1991).
Fujiyama, A., Miyanohara, A., Nozaki, C., Yoneyama, T., Ohtomo, N. and Matsubara, K., Cloning and structural analyses of hepatitis B virus DNAs, subtype adr.Nucleic Acids Res., 11, 4601–4610 (1983).
Kaneko, S., Miller, R., Feinstone, S., Unoura, M., Kobayashi, K., Hattori, N. and Purcell, R. H., Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay.Proc. Natl. Acad. Sci. USA., 86, 312–316 (1989).
Kaneko, S., Feinstone, S. M. and Miller, R. H., Rapid and sensitive method the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique.J. Clin. Microbiol., 27, 1930–1933 (1989).
Kim, Y. S. and Kang, H. S., Cloning and expression of hepatitis B virus surface antigen gene.Korean. Biochem. J., 17, 70–79 (1984).
Koh, K. C., Lee, H. S. and Kim, C. Y., Association of the core clustering mutations (codon 21–34) and the severity of chronic hepatitis B in Korean patients.Korean J. Intern. Med., 10, 87–93 (1995).
Kwok, S. and Higuchi, R., Avoiding false positives with PCR.Nature, 339, 237–238 (1989).
Liang, T. J., Hasegawa, K., Rimon, N., Wands, J. R. and Ben-Porath, E., A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis.N. Engl. J. Med., 324, 1705–1709 (1991).
Liang, T. J., Isseibacher, K. J. and Wands, J. R., Rapid identification of low level hepatitis B-related viral genome in serum.J. Clin. Invest., 84, 1367–1371 (1989).
Manzin, A., Salvoni, G., Bagnarelli, P., Menzo, S., Carloni, G. and Clement, M., A single step DNA extraction procedure for the detection of serum hepatitis B virus.J. Virol. Methods, 32, 245–253 (1991).
Norder, H., Hammas, B. and Magnius, L. O., Typing of hepatitis B virus genomes by a simplified polymerase chain reaction.J. Med. Virol., 31, 215–221 (1990).
Okamoto, H., Yotsomuto, S., Akahane, Y., Yamanaka, T., Miyazaki, Y., Sugai, F., Tanaka, T., Miyakawa, Y. and Mayumi M., Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen.J. Virol., 64, 1298–303 (1990).
Omata, M., Ehata, T., Yokosuka, O., Hosoda, K. and Ohto, M., Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis.N. Engl. J. Med., 324, 1699–1704 (1991).
Persing, D. H., Varmus, H. E. and Ganem, D., Inhibition of secretion of hepatitis B surface antigen by a related presurface polypeptide.Science, 234, 1388–1391 (1986).
Scully, L. J., Sung, H., Pennie, R. and Gill, P., Detection of hepatitis B virus DNA in the serum of Canadian hepatitis B surface antigen negative, anti-HBe positive individuals, using the polymerase chain reaction.J. Med. Virol., 44, 293–297 (1994).
Wright, T. L., Mamish, D., Combs, C., Kim, M., Donegan, E., Ferrell, L., Lake, J., Roberts, J. and Ascher, N. L., Hepatitis B virus and apparent fulminant non-A, non-B hepatitis.Lancet, 339, 952–955 (1992).
Zaaijer, H. L., ter Borg, F., Cuypers, H. T., Hermus, M. C. and Lelie, P. N., Comparison of methods for detection of hepatitis B virus DNA.J. Clin. Microbiol., 32, 2088–2091 (1994)
Zeldis, J. B., Lee, J. H., Mamish, D., Finegold, D. J., Sircar, R., Ling, Q., Knudsen, P. J., Kuramoto, I. K. and Mimms, L. T., Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction.J. Clin. Invest., 84, 1503–1508 (1989).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Jang, J.S., Lee, KJ. Simple and rapid identification of low level hepatitis B virus DNA by the nested polymerase chain reaction. Arch. Pharm. Res. 19, 469–474 (1996). https://doi.org/10.1007/BF02986013
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02986013