A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis

Authors
Marin, Maia Solange; Quintana, Silvina; Leunda, Maria Rosa; Recavarren, Mariana Ines; Pagnuco, Inti Anabela; Spath, Ernesto Juan; Perez, Sandra; Odeon, Anselmo Carlos
Publication Year
2016
Language
English
Format
article
Status
Published version
Description
Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle.
EEA Balcarce
Fil: Marin, Maia Solange. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Quintana, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Análisis Fares Taie; Argentina
Fil: Leunda, Maria Rosa. INTA. Estación Experimental Agropecuaria Balcarce; Argentina
Fil: Recavarren, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Análisis Fares Taie; Argentina
Fil: Pagnuco, Inti Anabela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Spath, Ernesto Juan. INTA. Estación Experimental Agropecuaria Balcarce; Argentina
Fil: Perez, Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina
Fil: Odeón, Anselmo Carlos. INTA. Estación Experimental Agropecuaria Balcarce; Argentina
Source
Journal of Virological Methods 227 : 14-22 (January 2016)
Subject
Ganado Bovino
Enfermedades de los Animales
Herpes Virus Bovino
PCR
Diagnóstico
Cattle
Animal Diseases
Bovine Herpesvirus
Diagnosis
Access level
Restricted access
License
Repository
INTA Digital (INTA)
Institution
Instituto Nacional de Tecnología Agropecuaria
OAI Identifier
oai:localhost:20.500.12123/4981