Hepatitis B virus (HBV) has been the most prevalent blood-borne pathogen wherein utero transmission has still not been properly managed. Recent practice guidelines suggested that an antiviral drug should be administered to third-trimester pregnancies with significant viremia (>2 × 105 IU/mL).
To develop a novel turbidity-based loop-mediated isothermal amplification (LAMP) coupled with heat treatment DNA extraction method that is a rapid, cost-effective, and feasible viral load assessment and could be applied to antenatal screening.
Primers and reagents were designed, turbidity-based platform and heat treatment method were added, and evaluated for optimal efficiency. Assay sensitivity was tested from serially diluted standard HBV DNA. Assay specificity was tested with six standard viral DNAs. Clinical samples were analyzed and the results were compared with those of quantitative polymerase chain reaction (qPCR) diagnostic records.
The optimized condition was 60°C with no betaine, 1.4 mM deoxyribonucleotide triphosphates (dNTPs) and 6 mM of MgSO4 for 60 min. The assay accurately detected samples with standard HBV DNA at >2 × 105 IU/mL in both distilled water and spiked serum. Results can be interpreted within 31.48 ± 1.41 min in real-time turbidimeter. The amplification is exclusively specific to HBV, but not with the other six human-specific viruses. Moreover, the assay showed comparable performance within 95% confidence interval to the previously developed HBV LAMP toward clinical specimens.
This newly developed method was accurate, affordable, and flexible to further implementation to large-scale third-trimester pregnancy screening.