Abstract
A digoxigenin-labelled RNA probe with a sensitivity of 800 50% tissue culture infectious dose (TCID50) was used to detect Hepatitis A Virus (HAV) in oysters. We studied the influence of extraction methodology on riboprobe detection. Oyster samples obtained by four methods of extraction and extraction-concentration were spiked with HAV (CF53 strain). There was no correlation between protein concentration and turbidity of samples, and anti-digoxigenin antibodies showed a non specific reaction. Background noise was independent of protein concentration and disappeared when HAV RNA isolation by phenol/chloroform extraction was introduced, but HAV RNA could not be detected by this technique. In the presence of Acid Guanidinium Thiocyanate (AGT), RNA from HAV suspension was detected following phenolic extraction with a detection threshold of 8.104 TCID50 of spotted virus. HAV detection in oyster extract by a digoxigenin-labelled riboprobe appeared useful in shellfish virology, at least for a primary screening of samples.
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