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

MARIN, M; QUINTANA, S; LEUNDA, M; RECAVARREN, M; PAGNUCO, I; SPATH, E; PEREZ, S. E; ODEÓN, A

JOURNAL OF VIROLOGICAL METHODS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2016 vol. 227 p. 14 - 22

0166-0934

Bovine herpesvirus types 1(BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim ofthis study was to develop a simple and reliable one-step real time PCR assaywith high resolution melting (HRM) analysis for the simultaneous detection anddifferentiation of BoHV-1 and BoHV-5. Optimization of assay conditions wasperformed with DNA from reference strains. Then, DNA from field isolates,clinical samples and tissue samples of experimentally infected animals werestudied by real time PCR-HRM. An efficient amplification of real time PCRproducts was obtained, and a clear melting curve and appropriate melting peaksfor both viruses were achieved in the HRM curve analysis for BoHVtype identification. BoHV was identified in allof the isolates and clinical samples, and BoHV types were properlydifferentiated. Furthermore, viral DNA was detected in12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivitycompared with virus isolation or conventional PCR. In this study, HRM was usedas a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovinealpha-herpesviruses DNA. Thus, this technique is an excellent tool fordiagnosis, research and epidemiological studies of these viruses in cattle.