Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR.
Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays. Every pathogen-specific assay had an analytical sensitivity of at least 100 genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers. The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics.
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Suedufer 10, 17493 Greifswald - Insel Riems, Germany. Electronic address: Kerstin.Wernike@fli.bund.de.
Kerstin
K
Wernike
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Suedufer 10, 17493 Greifswald - Insel Riems, Germany. Electronic address: Bernd.Hoffmann@fli.bund.de.
Bernd
B
Hoffmann
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Suedufer 10, 17493 Greifswald - Insel Riems, Germany. Electronic address: Martin.Beer@fli.bund.de.
Martin
M
Beer
D000199:Actins / Q000235:genetics; D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D014780:Viruses / Q000302:isolation & purification*
D000199:Actins
Bluetongue disease; Bovine viral diarrhoea; Epizootic haemorrhagic disease; Foot-and-mouth disease; Multiplex real-time PCR; Rift Valley fever