Ph.D.
Nguyen Thi Tuyet Nga

Title: Optimizing Primer Design and Nanopore Sequencing for the Detection of HBV Haplotype Variants

Name(s): Ha Khang Dat¹, Ngo Thi Nhung², Pham Thi Tuong Vy³, Nguyen Thi Tuyet Nga⁴
¹Pham Ngoc Thach University of Medicine
²University of Science, Vietnam National University Ho Chi Minh City
³Nong Lam University – Ho Chi Minh City
⁴Institute of Fundamental and Applied Sciences (IFAS), Duy Tan University.

The high genetic diversity of Hepatitis B virus (HBV) and the emergence of complex drug-resistant haplotypes pose major challenges to clinical management. Traditional sequencing methods (Sanger) and short-read next-generation sequencing (Illumina) have limitations in accurately phasing distant mutations within the same viral genome. This study aims to design, optimize in silico, and validate a workflow that employs specific PCR primers combined with Nanopore long-read sequencing to accurately detect drug resistant haplotypes of HBV in the polymerase gene. A dataset of 1.0277 complete HBV genome sequences (Genotypes A–J) was retrieved from GenBank. Multiple sequence
alignment (MSA) and Shannon entropy analysis were performed to identify highly conserved regions and variable regions containing drug resistance mutations (DRMs). Candidate primer pairs were designed using Primer3Plus, visualized in Clone Manager, and evaluated in silico for coverage using virtual PCR. The planned validation workflow includes two parallel branches from the same clinical sample: (1) conventional PCR (using three optimized 1.4 kb primer pairs) followed by Nanopore sequencing for haplotype analysis, and (2) qPCR (using independent short primers) for viral load quantification and sensitivity control. In silico analysis identified three optimal primer pairs with
100% theoretical coverage across all 11,014 surveyed sequences. These primers generate 1.4 kb amplicons spanning all major DRM positions in the polymerase gene. The expected findings indicate that the PCR–Nanopore workflow will achieve a detection rate comparable to qPCR in samples with moderate to high viral loads. Importantly, Nanopore sequencing is anticipated to enable accurate phasing of multidrug-resistant (MDR) haplotypes and detection of potentially novel variants that cannot be resolved by qPCR. This optimized workflow represents a powerful tool for both clinical medicine (supporting personalized therapy) and public health (epidemiological surveillance). It facilitates early detection of drug-resistant haplotypes in patients and can be applied to monitor the presence and transmission of HBV in the environment.

Biography of the presenting author

Dr. Nguyen Thi Tuyet Nga holds a Ph.D. in Life Sciences and Health from the University of Paris Saclay (2017). She is currently a Researcher and Lecturer at the Institute of Fundamental and Applied Sciences (IFAS), Duy Tan University. An active researcher specializing in molecular biology, clinical microbiology, and environmental biotechnology, she has led numerous research projects focusing on the optimization of diagnostic pipelines, including PCR/qPCR kit development and the application of Oxford Nanopore Technology for rapid pathogen identification and viral haplotype analysis. Her research portfolio spans diverse fields, from studying antimicrobial resistance in clinical and
wastewater samples to assessing environmental pollutants. Dr. Nga has published multiple high impact scientific papers in reputable international journals and is dedicated to advancing molecular diagnostic tools to support personalized medicine and public health surveillance. She bridges advanced biotechnological research with academic education in Vietnam.

Presenting author details

Full name: Nguyen Thi Tuyet Nga

Contact number: (+84) 869.178.868

Email: ngathituyetnguyen.20@gmail.com

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ORCID: https://orcid.org/0009-0009-3750-9103

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Category: Oral presentation