![]() qPCR was run with Primer 6 (Microsynth, 5-AGA GTA TCC ATC CGT AGT-3) and Primer 7 (Microsynth, 5-GGA GCT TGT GAA TTC TGG-3) at 100 nM with PowerUp SYBR Green Master Mix (Applied Biosystems, 5 L per 12.5 L reaction). It represents a convenient and rapid tool for future studies to investigate the clinical relevance of EHV-2 and EHV-5 in more detail. DNA was recovered by ethanol precipitation and the concentration was determined by absorption at 260 nm. Comparison of the Ct values of a selection of positive field samples showed only minimal differences between the singleplex and the multiplex assay. The here described multiplex rtPCR protocol allows sensitive and specific detection of EHV-2 and EHV-5. The lower limit of detection of the singleplex rtPCR was 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively. Melting curves and BLAST analysis of the sequences indicated specific detection of the viruses. Analytical sensitivity was assessed by standard curve generation of serial plasmid dilutions containing the respective target gene. Analytical specificity was determined by testing DNA of other herpesviruses by SYBR Green rtPCR and melting curve analysis, as well as Sanger sequencing of positive field samples. Microsynth is certified under ISO9001:2000 and ISO17025. Microsynth offer a wide range of molecular biology services and products including DNA sequencing, RNA synthesis, DNA synthesis, GMO testing, and Genotyping. Although their pathogenic potential is not yet fully understood, they appear to play a role in disease patterns like equine multinodular pulmonary fibrosis. In this study, a multiplex real-time PCR (rtPCR) was designed to detect DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5). Microsynth AG was founded in 1989 as the first private DNA synthesis company in Switzerland. ![]() Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are widely distributed in the equines.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |