Development of a Reagent Kit for the Quantitative Determination of Hepatitis B Virus (HBV) DNA in Clinical Material by PCR with Hybridization-Fluorescence Detection

Relevance. Hepatitis B virus (HBV) is one of the most common viral infections affecting people worldwide and can lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently 3% of the world's population are infected with hepatitis B virus and are at risk of developing life-threatening liver disease. Immunological and molecular biological methods of detection of HBV are currently used in laboratory diagnostics. The polymerase chain reaction (PCR) is currently the most sensitive method for the detection and quantification of HBV. HBV DNA quantification is widely used to monitor the antiviral treatment of HBV infection.

Aim. To develop a real-time PCR kit for the quantification of HBV DNA.

Materials and methods. A total of 200 plasma and serum samples positive and negative for HBV were used in the development. The performance of the developed kit was compared with the use of other commercially registered HBV diagnostic kits in Russia. Additionally, the nucleotide sequences of all existing virus genotypes analysed for the selection of primers using GeneBank system.

Results and discussion. Comparison analysis of the results of quantitative determination by real-time PCR in 200 clinical serum and blood plasma samples showed that the diagnostic sensitivity of the developed kit was 100% and specificity 100%. The primers developed specific to the POL gene region. The kit is capable of detecting all types of virus genotypes.

Conclusions. The developed reagent kit allows detection of hepatitis B virus and determination of its quantity within 70 minutes. In addition to a large number of genotypes and subgenotypes, the virus is characterized by mutational changes in the genome, which complicates its diagnosis and, as a consequence, the ongoing therapy with drugs. Conservative regions for primer and probe selection taken into account in the development, and the sequencing results obtained are applicable to all HBV genotypes. The reagent kit is designed to monitor HBV infected patients and will allow the analysis of different HBV viral loads.

1. Hepatitis B. World Health Organization. Available at: https://www.who.int/ru/news-room/fact-sheets/detail/hepatitis-b. Accessed: 24 May 2023 (In Russ).

2. Vilisova A.N. Development of a kit of reagents for detection and quantitative determination of hepatitis virus DNA by polymerase chain reaction. Medicine and pharmacy, past, present, future: IV All-Russian scientific-practical conference with international participation, devoted to the Year of teacher and mentor; 21 Apr 2023; OrekhovoZuyevo, Russia. EPD GGTU; 2023. P. 43-47. (In Russ).

3. Ganem D., Prince A. M. Hepatitis B Virus Infection — Natural History and Clinical Consequences. New England Journal of Medicine. 2004;350(11):1118–1129. doi: 10.1056/nejmra031087

4. Hu J., Seeger C. Hepadnavirus Genome Replication and Persistence. Cold Spring Harbor perspectives in medicine. 2015(7):1–16. doi: 10.1101/cshperspect.a021386

5. Venkatakrishnan B., Zlotnick A. The Structural Biology of Hepatitis B Virus: Form and Function. Annual review of virology. 2016;3(1):429–451. doi: 10.1146/annurev-virology-110615-042238

6. Hui L., Junjun C., Usha V., et al. Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis. PLoS Pathog. 2021;17(11):1–34. doi: 10.1371/journal.ppat.1010057

7. Eren E., Watts N. R., Dearborn A. D., et al. Structures of Hepatitis B Virus Coreand e-Antigen Immune Complexes Suggest Multi-point Inhibition. Structure. 2018;26(10):1314– 1326. doi: 10.1016/j.str.2018.06.012

8. Selzer L., Zlotnick A. Assembly and Release of Hepatitis B Virus. Cold Spring Harbor perspectives in medicine. 2015;5(12):1–17. doi: 10.1101/cshperspect.a021394

9. Teng C-F., Wu H-C., Su I-J., Jeng L-B. Hepatitis B Virus Pre-S Mutants as Biomarkers and Targets for the Development and Recurrence of Hepatocellular Carcinoma. Viruses. 2020;12(9):1–13. doi: 10.3390/v12090945

10. Mendenhall M.A., Hong X., Hu J. Hepatitis B Virus Capsid: The Core in Productive Entry and Covalently Closed Circular DNA Formation. Viruses. 2023;15(3):1–11. doi: 10.3390/v15030642

11. Acute hepatitis B (HB) in adults [Internet]. Available at: https://cr.minzdrav.gov.ru/schema/672_1. Accessed: 24 May 2023 (In Russ).

12. Zaitsev IA., Novak IM., Zaitseva OYe., et al. Significance of hepatitis B virus genotypes in clinical practice. Current Infectiology. 2019;7(2):63–70. (In Russ). doi: 10.22141/2312413x.7.2.2019.161150

13. CJSC «EKOlab». PCR diagnostic department – CovidEK Extract. Available at: https://ekolab.ru/catalog/laboratornaya-diagnostik/otdelenie-ptsr/kovidek-ekstrakt/. Accessed: 24 May 2023 (In Russ).

14. Basic Local Alignment Search Tool. National Center for Biotechnology Information. Available at: https://blast.ncbi.nlm.nih.gov/Blast.cgi. Accessed: 24 May 2023.

15. Zhigaleva O.N., Mardanly S.G., Gashenko T.Yu., et al. Development of a reagent kit for qualitative detection of Hepatitis C virus (HCV) RNA in clinical material by real-time PCR with hybridization-fluorescence detection. Klinicheskaya Laboratornaya Diagnostika (Russian Clinical Laboratory Diagnostics). 2023; 68(7): 437-442 (in Russ.) doi. org:10.51620/0869-2084-2023-68-7-437-442.

16. Zhigaleva O.N., Mardanly S.G., Gashenko T.Yu., et al. Development of a reagent kit for qualitative detection of Hepatitis B virus (HBV) DNA in clinical material by real-time PCR with hybridization-fluorescence detection. Klinicheskaya Laboratornaya Diagnostika (Russian Clinical Laboratory Diagnostics). 2023; in press.