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©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections, rt269L and rt269I
Kijeong Kim, Yu-Min Choi, Dong Hyun Kim, Junghwa Jang, Won Hyeok Choe, Bum-Joon Kim
Kijeong Kim, Department of Microbiology, College of Medicine, Chung-Ang University, Seoul 06974, South Korea
Yu-Min Choi, Dong Hyun Kim, Junghwa Jang, Bum-Joon Kim, Department of Microbiology and Immunology, College of Medicine, Seoul National University, Seoul 03080, South Korea
Won Hyeok Choe, Department of Internal Medicine, Konkuk University School of Medicine, Seoul 05030, South Korea
Bum-Joon Kim, Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul 03080, South Korea
Bum-Joon Kim, Liver Research Institute, College of Medicine, Seoul National University, Seoul 03080, South Korea
Bum-Joon Kim, Seoul National University Medical Research Center, Seoul 03080, South Korea
Author contributions: Kim K and Kim BJ contributed to study conception and design, and designed and performed experiments; Choe WH contributed to collection of clinical data; Kim K, Choi YM, Kim DH, Jang J, Choe WH, and Kim BJ contributed to data acquisition, data analysis and interpretation; Kim K, Choi YM, Choe WH, and Kim BJ contributed to writing of article, editing, reviewing and final approval of article.
Supported by the National Research Foundation of Korea, No. 2022R1A2B5B01001421; and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, the Ministry of Health & Welfare, Republic of Korea, No. HI22C0476.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board at Seoul National University Hospital.
Informed consent statement: Informed consent was waived because of the retrospective nature of the study and the analysis used anonymous clinical data.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
https://creativecommons.org/Licenses/by-nc/4.0/ Corresponding author: Bum Joon Kim, PhD, Professor, Department of Microbiology and Immunology, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul 03080, South Korea.
kbumjoon@snu.ac.kr
Received: October 14, 2022
Peer-review started: October 14, 2022
First decision: January 3, 2023
Revised: January 13, 2023
Accepted: February 27, 2023
Article in press: February 27, 2023
Published online: March 21, 2023
Processing time: 153 Days and 23.8 Hours
BACKGROUND
The presence of two distinct hepatitis B virus (HBV) Pol RT polymorphisms, rt269L and rt269I, could contribute to the unique clinical or virological phenotype of HBV genotype C2. Therefore, a simple and sensitive method capable of identifying both types in chronic hepatitis B (CHB) patients infected with genotype C2 should be developed.
AIM
To develop a novel simple and sensitive locked nucleic acid (LNA)-real time-polymerase chain reaction (RT-PCR) method capable of identifying two rt269 types in CHB genotype C2 patients.
METHODS
We designed proper primer and probe sets for LNA-RT-PCR for the separation of rt269 types. Using synthesized DNAs of the wild type and variant forms, melting temperature analysis, detection sensitivity, and endpoint genotyping for LNA-RT-PCR were performed. The developed LNA-RT-PCR method was applied to a total of 94 CHB patients of genotype C2 for the identification of two rt269 polymorphisms, and these results were compared with those obtained by a direct sequencing protocol.
RESULTS
The LNA-RT-PCR method could identify two rt269L and rt269I polymorphisms of three genotypes, two rt269L types [‘L1’ (WT) and ‘L2’] and one rt269I type (‘I’) in single (63 samples, 72.4%) or mixed forms (24 samples, 27.6%) in 87 (92.6% sensitivity) of 94 samples from Korean CHB patients. When the results were compared with those obtained by the direct sequencing protocol, the LNA-RT-PCR method showed the same results in all but one of 87 positive detected samples (98.9% specificity).
CONCLUSION
The newly developed LNA-RT-PCR method could identify two rt269 polymorphisms, rt269L and rt269I, in CHB patients with genotype C2 infections. This method could be effectively used for the understanding of disease progression in genotype C2 endemic areas.
Core Tip: Hepatitis B virus (HBV) genotype C2 infections have distinct clinical or virological traits, including a higher risk of hepatocellular carcinoma, lower response rate to interferon or prolonged hepatitis B e antigen-positive phase. We recently reported that the presence of two HBV Pol RT polymorphisms, rt269L and rt269I, contributed to unique traits of HBV genotype C2. Here, instead of time- or labor-consuming direct sequencing, we developed a new locked nucleic acid (LNA)-real time-polymerase chain reaction (RT-PCR) method for the separation between rt269L (L1 and L2) and I type from Korean chronic hepatitis B patients of genotype C2. The newly developed LNA-RT-PCR could be effectively used for the understanding of epidemiology and disease progression in genotype C2 endemic areas.
