Pectin methylesterase (PME) was extracted from strawberries (Fragaria ananassa, cv Elsanta) and purified by affinity chromatography on a CNBr-Sepharose 4B-PME-inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molecular mass, pI, and kinetic parameters of strawberry PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified strawberry PME could be described by a fractional-conversion model. Purified strawberry PME is much more stable toward high-pressure treatments in comparison to those from oranges and bananas.

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We developed a novel real-time PCR assay that simultaneously evaluates eleven major nucleos(t)ide antiviral (NA) drug-resistance mutations (mt) in chronic hepatitis B patients (CHB), including L180M, M204I/V, and V207M (lamivudine (LMV) resistance), N/H238A/T (Adefovir (ADF) resistance) which are circulating in Vietnam; T184G/L, S202I, M250V (entecavir (ETV) resistance), and A194T (tenofovir resistance) which have been recently reported in several studies across the globe. We detected drug-resistant mt in HBV samples using our predesigned panel of allele-specific locked-nucleic acid (LNA) probes. Our assay had a high sensitivity of 5% in a low HBV DNA population of ≥5 ×10 3 IU/mL and was validated in a cohort of 130 treatment-naive children and 98 NA-experienced adults with CHB. Single-point mt for LMV- and ADF-resistance were detected in 57.7% and 54.1% of the child and adult samples, respectively, with rtV207M (42.3%, children; 36.7%, adults) and rtN238T/A (15.4%, children; 16.3, adults) being the most frequent mt in these populations. Multiple-point mt including rtL180M-rtM204V- rtN238A and rtL180M-rtM204I were only identified in two children, resulting in LMV-ADF-resistance and reduced ETV susceptibility. In conclusion, this assay accurately identified the mt profile of children (98.4%) and adults (91.2%) with CHB, which is comparable to established methods. This fast and sensitive screening method can be used for the detection of major NA-resistant mt circulating in developing countries, as well as providing a model for the development of similar mt-detection assays, especially for use in non-hospitalised patients who need their results within half a day before starting treatment.

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