HBV DNA Test Among Blood Donations May Require Two Amplification Targets

  • Chao Liu
  • Le Chang
  • Lunan WangEmail author
Original Article


To analyze the risk and reason of false-negative HBV DNA results of NAT reagents among blood donations of China and discuss the necessity of two amplification targets for HBV DNA tests among donations. In this study, samples that showed discordant results on two commercially available assay platforms were further detected by established in-house methods based on conserved regions of the HBV genome. The HBV concentration of these samples was determined using two commercially available reagents. The samples with high titers of HBV were detected by an in-house method. The samples showing high Ct differences between two regions in the in-house method were further sequenced and aligned with primers and probes. The results showed that the established method has a good detection performance. The mismatch between reverse primers and sample sequences led to decreased detection capacity of S and C regions by the in-house method, but it could be compensated by another region. Among the false-negative samples detected by commercial reagents, most were because of low titers; however, there were 7 samples with HBV DNA concentrations much higher than the LOD of the commercial reagents, which may be due to mismatch of the primer or probe. This study highlights the potential risk of HBV false-negative detection by commercial NAT reagents. The dual-target assay may be helpful for HBV screening and reduce the risk of false-negative detection.


Hepatitis B virus Nucleotide acid test Dual-target False-negative Transmission risk 



The authors thank the staff at the participating blood banks who are engaged in this study.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no competing interest.

Ethical Statement

The study was approved by the institutional review board of the National Center for Clinical Laboratories. Written informed consent was obtained from all subjects participating in this research.


  1. 1.
    Custer B, Sullivan SD, Hazlet TK, Iloeje U, Veenstra DL, Kowdley KV (2004) Global epidemiology of hepatitis B virus. J Clin Gastroenterol 38:S158–S168CrossRefGoogle Scholar
  2. 2.
    Liang X, Bi S, Yang W, Wang L, Cui G, Cui F, Zhang Y, Liu J, Gong X, Chen Y, Wang F, Zheng H, Wang F, Guo J, Jia Z, Ma J, Wang H, Luo H, Li L, Jin S, Hadler SC, Wang Y (2009) Epidemiological serosurvey of hepatitis B in China—declining HBV prevalence due to hepatitis B vaccination. Vaccine 27:6550–6557CrossRefGoogle Scholar
  3. 3.
    Liu GC, Sui GY, Liu GY, Zheng Y, Deng Y, Gao YY, Wang L (2013) A Bayesian meta-analysis on prevalence of hepatitis B virus infection among Chinese volunteer blood donors. PLoS ONE 8:e79203CrossRefPubMedCentralGoogle Scholar
  4. 4.
    Liu C, Chang L, Ji H, Guo F, Zhang K, Lin G, Zhang R, Li J, Wang L (2016) Prevalence of HBV DNA among 20 million seronegative blood donations in China from 2010 to 2015. Sci Rep. 6:36464CrossRefPubMedCentralGoogle Scholar
  5. 5.
    Echevarría JM, Avellón A (2006) Hepatitis B virus genetic diversity. J Med Virol 78(Suppl):S36–S42CrossRefGoogle Scholar
  6. 6.
    Girones R, Miller RH (1989) Mutation rate of the hepadnavirus genome. Virology 170:595–597CrossRefGoogle Scholar
  7. 7.
    Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, Tyrrell DL (1996) Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 24:714–717Google Scholar
  8. 8.
    Dos Santos MIMA, Pacheco SR, Stocker A, Schinoni MI, Paraná R, Reis MG, Silva LK (2017) Mutations associated with drug resistance and prevalence of vaccine escape mutations in patients with chronic hepatitis B infection. J Med Virol 89:1811–1816CrossRefGoogle Scholar
  9. 9.
    Lindh M, Hannoun C, Malmström S, Lindberg J, Norkrans G (2006) Lamivudine resistance of hepatitis B virus masked by coemergence of mutations in probe region of the COBAS AMPLICOR assay. J Clin Microbiol 44:2587–2589CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Liu C, Chang L, Jia T, Guo F, Zhang L, Ji H, Zhao J, Wang L (2017) Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets. Virol J 14:94CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Chudy M, Weber-Schehl M, Pichl L, Jork C, Kress J, Heiden M, Funk MB, Nübling CM (2012) Blood screening nucleic acid amplification tests for human immunodeficiency virus Type 1 may require two different amplification targets. Transfusion 52:431–439CrossRefGoogle Scholar
  12. 12.
    Shah K, Ragupathy V, Saga A, Hewlett I (2016) High sensitivity detection of HIV-1 using two genomic targets compared with single target PCR. J Med Virol 88:1092–1097CrossRefGoogle Scholar
  13. 13.
    Tsoi WC, Lelie N, Lin CK (2013) Enhanced detection of hepatitis B virus in Hong Kong blood donors after introduction of a more sensitive transcription-mediated amplification assay. Transfusion 53:2477–2488CrossRefGoogle Scholar
  14. 14.
    Taira R, Satake M, Momose S, Hino S, Suzuki Y, Murokawa H, Uchida S, Tadokoro K (2013) Residual risk of transfusion-transmitted hepatitis B virus (HBV) infection caused by blood components derived from donors with occult HBV infection in Japan. Transfusion 53:1393–1404CrossRefGoogle Scholar
  15. 15.
    Larralde O, Dow B, Jarvis L, Davidson F, Petrik J (2013) Hepatitis B escape mutants in Scottish blood donors. Med Microbiol Immunol 202:207–214CrossRefGoogle Scholar
  16. 16.
    Allain JP, Mihaljevic I, Gonzalez-Fraile MI, Gubbe K, Holm-Harritshøj L, Garcia JM, Brojer E, Erikstrup C, Saniewski M, Wernish L, Bianco L, Ullum H, Candotti D, Lelie N, Gerlich WH, Chudy M (2013) Infectivity of blood products from donors with occult hepatitis B virus infection. Transfusion 53:1405–1415CrossRefGoogle Scholar
  17. 17.
    Andonov A, Osiowy C, Borlang J, Swidinsky K (2016) Sequence variability of the Cobas taqman assay target region impacts accurate HBV DNA detection. Vox Sang 111:S58Google Scholar

Copyright information

© Indian Society of Hematology and Blood Transfusion 2019

Authors and Affiliations

  1. 1.National Center for Clinical LaboratoriesBeijing Hospital, National Center of GerontologyBeijingPeople’s Republic of China
  2. 2.Beijing Engineering Research Center of Laboratory MedicineBeijing HospitalBeijingPeople’s Republic of China
  3. 3.Graduate School, Peking Union Medical CollegeChinese Academy of Medical SciencesBeijingPeople’s Republic of China

Personalised recommendations