METHODS: Here, we report a simple iron flocculation method for concentrating viruses in water, together with a newly-developed hydrolysis probe quantitative RT-qPCR method for the detection and quantification of TiLV.
RESULTS: The RT-qPCR method designed to target a conserved region of the TiLV genome segment 9 has a detection limit of 10 viral copies per µL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 ± 3.3% of the virus from water samples spiked with viral cultures. Tilapia and water samples were collected for use in the detection and quantification of TiLV disease during outbreaks in an open-caged river farming system and two earthen fish farms. TiLV was detected from both clinically sick and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms (i.e., river water samples from affected cages (8.50 × 103 to 2.79 × 105 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 × 103 to 3.53 × 104 copies/L)). By contrast, TiLV was not detected in fish or water samples collected from two farms that had previously experienced TiLV outbreaks and from one farm that had never experienced a TiLV outbreak. In summary, this study suggests that the eRNA detection system using iron flocculation, coupled with probe based-RT-qPCR, is feasible for use in the concentration and quantification of TiLV from water. This approach may be useful for the non-invasive monitoring of TiLV in tilapia aquaculture systems and may support evidence-based decisions on biosecurity interventions needed.
METHODS: The fruit powder was macerated using methanol and then partitioned by hexane, ethyl acetate, n-butanol, and water. The fractions were chromatographed on the column chromatography and incorporated with TLC and recrystallization to give pure compounds. The structures of isolated compounds were determined by UV-Visible, FT-IR, MS, proton (1H-NMR), carbon (13C-NMR), and 2D-NMR techniques encompassing HMQC and HMBC spectra. The compounds were evaluated for their ACE inhibitory activity, and the strongest compound was determined by the kinetics enzyme inhibition.
RESULTS: Based on the spectral data, the isolated compounds were determined as 6,4-dihydroxy-4-methoxybenzophenone-2-O-β-D-glucopyranoside (1), 4,4'-dihydroxy-6-methoxybenzophenone-2-O-β-D-glucopyranoside (2) and mangiferin (3). IC50 values of the isolated compounds 1, 2 and 3 were 0.055, 0.07, and 0.025 mM, respectively.
CONCLUSION: The three compounds have ACE inhibitor and mangiferin demonstrated the best ACE inhibitory activity with competitive inhibition on ACE with the type of inhibition kinetics is competitive inhibition.