Abstract
Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), 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, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 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 fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).
Collomb, J;
Finance, C;
Alabouch, S;
[1]
Laporte, J
[2]
- Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France)
- Station de Virologie et d'Immunologie Moleculaires, INRA, Jouy-en-Josas (France)
Citation Formats
Collomb, J, Finance, C, Alabouch, S, and Laporte, J.
Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization.
Austria: N. p.,
1992.
Web.
doi:10.1007/BF01309626.
Collomb, J, Finance, C, Alabouch, S, & Laporte, J.
Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization.
Austria.
https://doi.org/10.1007/BF01309626
Collomb, J, Finance, C, Alabouch, S, and Laporte, J.
1992.
"Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization."
Austria.
https://doi.org/10.1007/BF01309626.
@misc{etde_6444106,
title = {Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization}
author = {Collomb, J, Finance, C, Alabouch, S, and Laporte, J}
abstractNote = {Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), 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, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 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 fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).}
doi = {10.1007/BF01309626}
journal = []
volume = {125:1-4}
journal type = {AC}
place = {Austria}
year = {1992}
month = {Jan}
}
title = {Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization}
author = {Collomb, J, Finance, C, Alabouch, S, and Laporte, J}
abstractNote = {Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), 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, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 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 fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).}
doi = {10.1007/BF01309626}
journal = []
volume = {125:1-4}
journal type = {AC}
place = {Austria}
year = {1992}
month = {Jan}
}