Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization.
Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes, were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods.
Collomb
J
J
Laboratoire de Microbiologie Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques, Université de Nancy I, France.
Finance
C
C
Alabouch
S
S
Laporte
J
J
D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003332:Coronaviridae / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics; D005243:Feces / Q000382:microbiology; D006801:Humans; D007124:Immunoenzyme Techniques; D010761:Phosphorus Radioisotopes; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D004279:DNA, Viral; D010761:Phosphorus Radioisotopes; D012367:RNA, Viral