Published online Mar 21, 2023. doi: 10.3748/wjg.v29.i11.1721
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
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.
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.
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.
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).
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.