Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference.
A novel two-step real-time RT-PCR assay using SYBR Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5'-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.
Young
N J
NJ
Department of Pathology and Infectious Diseases, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK. nyoung@rvc.ac.uk
Thomas
C J
CJ
Collins
M E
ME
Brownlie
J
J
D020121:5' Untranslated Regions; D000818:Animals; D052160:Benzothiazoles; D001769:Blood / Q000821:virology*; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000821:virology; D002417:Cattle; D017934:Coronavirus; D017931:DNA Primers; D003959:Diamines; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D019590:Nucleocapsid Proteins / Q000235:genetics; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D014776:Virus Cultivation
D020121:5' Untranslated Regions; D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D019590:Nucleocapsid Proteins; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I