High Resolution Melting (HMR) como nuevo metodo para la deteccionn simultanea y discriminacion de los herpesvirus bovinos tipo 1 (BoHV-1) y tipo 5 (BoHV-5).

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

Tucuman

Jornada; XX Reunion Cientifico Tecnica de Laboratorios de Diangostico Veterinario; 2014

AALDV

Introduction Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are two closely related alpha-herpesviruses that infect cattle. Despite their high similarity, differentiation among BoHV-1 and BoHV-5 is not achieved by classical virological techniques. Thus, virus isolates and samples collected from affected animals must be analyzed using sensitive and reliable molecular methods. High resolution melting (HRM) analysis is a recently developed molecular technique. The development of HRM curve analysis, as an extension to Real time PCR, is based on the determination of changes in fluorescence as a result of melting double-stranded PCR products in response to temperature increase. The melting temperature (Tm) and the characteristic shape in the melting curve profile of the amplified products are highly dependent on the nucleotide sequence. There are no reports on the use of Real time PCR with HRM for the identification of bovine alpha-herpesviruses. The aim of this study was to develop a simple and reliable one-step Real time PCR assay with HRM analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5.   Materials and methods Reference strains LA (BoHV-1.1), Cooper (BoHV-1.1), N569 (BoHV-5a) and A663 (BoHV-5b), Argentinean field isolates (10 BoHV-1 and 1 BoHV-5 strains), 5 clinical samples in which BoHV DNA was detected by conventional PCR and 45 samples from the respiratory tract of experimentally infected animals were studied. DNA was extracted from viral isolates or tissue samples using a commercial kit (Qiagen Inc., Valencia, CA, USA). To identify the most effective set of primers for detecting and differentiating BoHV-1 and BoHV-5, three primers pairs were evaluated. The sequences of BoHV-1 and BoHV-5 obtained from GenBank database, corresponding to the amplified fragments, were aligned and compared by use of MAFFT multiple sequence alignment program. Assay conditions were optimized using varying primer concentrations, annealing temperature and time. Real Time PCR assays were carried out in a Rotor Gene Q thermocycler, in a final volume of 20 ìl using EvaGreen as intercalating fluorescent dye (KAPA HRM FAST). The cycling program consisted of an initial denaturation of 3 minutes at 95°C and 50 cycles of 15 seconds at 95°C, 15 seconds at 58°C and 20 seconds at 72°C. After amplification, a HRM curve analysis was performed and BoHV identification was performed using Rotor Gene Q software, version 1.7.94. The sensitivity and specificity of the assay was determined after amplification and genotyping of DNA from isolates and tissue samples. Moreover, the Real Time PCR-HRM was compared to virus isolation and conventional PCR (Campos et al., 2009) techniques using tissue samples from experimentally infected animals. Concordance between the three methodologies was assessed using Cohen?s Kappa coefficient. The difference among coefficients was calculated using the Chi-square test (P