Rapid detection and differentiation of bovine herpesvirus 1 and 5 glycoprotein C gene in clinical specimens by multiplex-PCR.
A multiplex polymerase chain reaction (multiplex-PCR) to detect and differentiate bovine herpesvirus 1 (BoHV-1) and 5 (BoHV-5) was developed using primers for the gene sequence that encodes the glycoprotein C. The technique was assessed against the BoHV-1 and BoHV-5 cell culture adapted strains, and clinical samples collected from animals with clinical signs of BoHV-1 (n = 10) or BoHV-5 (n = 7) infection and with diagnosis confirmed by virus isolation in cell culture and semi-nested PCR. Fifteen clinical samples from asymptomatic animals were included as control group. For the evaluation of the amplifiability of the extracted nucleic acid from clinical specimens was included a bovine internal control that amplified a 626 bp fragment of the ND5 gene present in the bovine mitochondrial DNA. For DNA extraction, a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate methods was used. The specificity of the BoHV-1 and BoHV-5 amplicons from standard strains were confirmed by sequence analysis. All the positive clinical samples for BoHV included in this study were characterized as BoHV-1 or BoHV-5 by the difference in length of the amplified product visualized in a agarose gel (354 bp size for BoHV-1, and 159 bp for BoHV-5). The internal control was amplified in all clinical specimens. Non-specific reactions were not observed when the multiplex-PCR was assessed with other viruses (bovine viral diarrhea virus and rabies virus) and BoHV-negative clinical samples from fetuses and adult cattle obtained from a slaughterhouse.
Claus
Marlise Pompeo
MP
Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina (UEL), Londrina/PR, Brazil.
Alfieri
Alice Fernandes
AF
Folgueras-Flatschart
Aurea Valadares
AV
Wosiacki
Sheila Rezler
SR
Médici
Kerlei Cristina
KC
Alfieri
Amauri Alcindo
AA
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000821:virology; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D018792:Encephalitis, Viral / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D007242:Herpesvirus 1, Bovine / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D025881:Herpesvirus 5, Bovine / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007241:Infectious Bovine Rhinotracheitis / Q000175:diagnosis / Q000821:virology; D008590:Meningoencephalitis / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000235:genetics*
D004279:DNA, Viral; D014759:Viral Envelope Proteins; C038535:glycoprotein gC, herpes simplex virus type 1