Hepatitis B remains one of the world’s most consequential viral infections. The virus chronically infects a substantial share of the global population, and long-standing infection can progress to cirrhosis and liver cancer. The burden falls disproportionately on low-resource settings, precisely where sophisticated diagnostic infrastructure is scarcest. Quantitative PCR, the molecular gold standard, demands expensive thermocyclers, fluorescence optics, trained personnel and reliable supply chains, which confines it to urban reference laboratories. Serological rapid tests are cheap and widely deployed, but they detect antibodies or viral antigens rather than viral DNA, and their performance can falter early in infection or in immunocompromised patients. The gap between what is technically possible and what is practically available has long motivated the search for alternative amplification chemistries.

Polymerase spiral reaction belongs to a family of isothermal amplification methods that dispense with the repeated heating and cooling cycles of conventional PCR. Instead of a pair of standard primers, PSR uses a specially designed set of four primers that recognize six distinct regions on the target sequence. The primers carry self-complementary tails, so the amplicons they generate fold into structures that present single-stranded loops resembling the original target. A strand-displacing polymerase then primes new synthesis from these loops, setting off a self-sustaining spiral of extension and displacement. Because the reaction never needs to melt double-stranded DNA thermally, it runs at a single fixed temperature, in this case 63 degrees Celsius, achievable with a simple water bath or heating block.

The research team, led by Prerana Sharan and Praveen Rai of Nitte University Centre for Science Education and Research, with collaborators at KS Hegde Medical Academy, AJ Institute of Medical Sciences and the National Institute of Biologicals, targeted a specific region of the HBV S gene, which encodes the viral surface antigen. Primer design is the critical step in any isothermal assay: the primers must bind tightly enough to initiate amplification but not so promiscuously that they generate signal from unrelated sequences. After systematic optimization of reaction temperature, duration, magnesium concentration and primer amounts, the assay settled on 63 degrees for 60 minutes as the condition that maximized amplification efficiency while preserving specificity.

The visual readout relies on hydroxy naphthol blue, a metallochromic dye that changes hue depending on the amount of free magnesium in the reaction mixture. As the polymerase incorporates nucleotides, it consumes magnesium ions, and the accumulating amplicons shift the dye from violet to blue. An operator can therefore distinguish positive from negative reactions by naked eye within the hour, without fluorescence detectors, gel electrophoresis or any optical instrumentation. This dye-based approach has been used with other isothermal methods, but its combination with PSR for HBV is what the new study demonstrates.

Analytical performance was striking. Serial dilutions of quantified HBV plasmid DNA established a detection limit of 5.04 copies per reaction, a figure that places the assay among the most sensitive isothermal methods reported for this virus. Specificity testing showed no cross-reactivity with other hepatitis-causing viruses, including hepatitis C virus, or with a panel of unrelated pathogens, indicating that the primer set discriminates reliably between HBV and genetically distinct organisms that might co-circulate in the same patient.

Clinical evaluation followed. The team tested 85 samples, including 45 previously screened as HBV-positive by serological assay. Against the reference results, the PSR assay achieved 100 percent sensitivity and 100 percent specificity, a perfect concordance rate in the evaluated set. The authors note that the assay outperformed conventional PCR in the clinical samples they examined, although they acknowledge that the sample size, while adequate for a proof of concept, will need expansion in larger multi-center trials before the assay can be deployed at scale.

The study was approved by the Institutional Ethics Committee of Nitte Deemed to be University and used anonymized residual clinical samples, with the requirement for informed consent waived. The work was supported by a Faculty Research Grant from Nitte Deemed to be University. The article is published open access under a Creative Commons Attribution 4.0 license, and the authors declare no competing interests.

The implications extend beyond hepatitis B. The same PSR chemistry, coupled with HNB or related dyes, could in principle be adapted to other viral targets by redesigning the primer set, offering a modular platform for point-of-care molecular diagnosis. The authors frame their assay as a tool for early surveillance and detection, particularly aimed at healthcare in poor-resource settings, where the combination of isothermal amplification and dye-based visual readout eliminates the need for complex instruments. Whether the assay can maintain its analytical performance outside the controlled environment of a research laboratory, and whether it can be integrated into routine screening programs, are questions that larger validation studies will need to address.

For now, the study adds to a growing body of work demonstrating that molecular diagnosis need not be confined to centralized facilities. A reaction that runs at a single temperature, completes in an hour, and announces its result through a color change visible to the naked eye represents a meaningful step toward diagnostics that meet the virus where it actually circulates. As the authors conclude, the HBV-PSR assay could potentially serve as a point-of-care diagnostic tool for HBV detection, with the goal of early surveillance and detection focused on healthcare in resource-poor settings.

Subject of Research: Virology

Article Title: Colorimetric polymerase spiral reaction (PSR) assay for the rapid detection of hepatitis B virus

Article References: Sharan, P., Bhandary, R., Rao, C., Maiti, B., Kumar, B. K., Kumar, A., & Rai, P. (2026). Colorimetric polymerase spiral reaction (PSR) assay for the rapid detection of hepatitis B virus. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14058-8

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14058-8

Keywords: Colorimetric, polymerase, spiral, reaction, assay, rapid, detection, hepatitis, virus, scientific research