科研之友
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摘要
Outbreaks of hepatitis A virus (HAY) infections are most frequently associated with the consumption of contaminated oysters. A rapid and selective concentration method is necessary for the recovery of HAY from contaminated oysters prior to detection using PCR. In this study, ricin extracted from castor beans (Ricinus communis) was tested as an alternative to antibody used in immunomagnetic separation while concentrating HAY prior to its detection using reverse transcription PCR. Initially, the extracted proteins from castor beans were fractionated into 13 fractions by gel filtration chromatography. Pretreatment of different protein fractions showed a variation in binding of HAY viral protein (VP) 1 to oyster digestive tissue in the range of 25.9 to 63.9%. The protein fraction, which caused the highest reduction in binding of VP1 to the tissue, was identified as ricin A by quadrupole time-of-flight mass spectrometry. Ricin A could significantly inhibit binding of VP1 to the tissue with a 50% inhibitory concentration of 4.5 mu g/ml and a maximal inhibitory concentration of 105.2%. The result showed that the rate of inhibition of HAY binding to tissue was higher compared to the rate of ricin itself binding to HAY (slope: 0.0029 versus 0.00059). However, ricin A concentration showed a higher correlation to the relative binding of ricin itself to HAY than the inhibition of binding of HAY to the tissue (coefficient of determination, R-2: 0.9739 versus 0.6804). In conclusion, ricin A linked magnetic bead separation combined with reverse transcription PCR can successfully detect HAY in artificially seeded oyster digestive tissue up to a 10(-4) dilution of the virus stock (titer: 10(4) 50% tissue culture infective dose per ml).
- 出版日期2015-5
