Detection of bovine enteric coronavirus in clinical specimens by hybridization with cDNA probes

Abstract

Molecular hybridization, previously optimized for purified bovine coronavirus (BCV), was adapted for detection of virus in clinical specimens. For this purpose, the accuracy of the existing tests had to be improved and suitable means for removal of extraneous molecules had to be established. Six radioactive probes were used to obtain adequate detection signals. These probes, containing the complete N and E1 gene sequences and other sequences, hybridized to about 1 4 of the total length of the viral RNA. Genomic RNA could be detected after direct spotting of samples, but prior Freo-nextraction or centrifugation of specimens on a cushion of sucrose improved considerably the positive identification of virus containing samples. RNA detection in positive clinical specimens was significantly better by hybridization than immunological detection of BCV by ELISA, although differences were slight after two passages of the virus on HRT-18 cell monolayers. Consequently, the reliability of positive and negative test results in hybridization tests on Freon extracted specimens was better than in ELISA. However, results after extraction with other organic solvents were inferior. The accuracy of ELISA was surpassed by hybridization assays. Background signals, due to vector homology were found to be negligible in all the samples analyzed.