Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus.

Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus. Rev Argent Microbiol. 2020 Sep 10;: Authors: Rojas MF, König GA, Vagnozzi AE, Vera FS, Scolaro LA, Craig MI Abstract A previous sequence analysis of a US5 gene fragment of infectious laryngotracheitis virus (ILTV) performed in an Argentinian epidemiological study allowed to differentiate between wild and vaccine strains. This analysis also defined five ILTV haplotypes with specific variations at positions 461, 484, 832, 878 and 894 of the US5 gene. This characterization of viral strains may also be accomplished using the High-Resolution Melting Analysis (HRMA), which has been described as an effective, fast and sensitive method to detect mutations in PCR products. In the present study, an HRM protocol was developed with the aim of characterizing the circulating ILTV strains in Argentina. The specificity of this tool was confirmed in different DNA diluents, without interference from heterologous DNA or other cellular metabolites. Additionally, the salt concentration in the elution buffer used for DNA extraction did not alter the curve profiles. Higher concentrations of DNA (Ct≅26.0) displayed well-defined curve profiles, whereas lower concentrations (Ct≅32.5) exhibited more heterogeneous curves. The HRMA showed 97.49% concordance with the reference technique, i.e., sequencing. The HRM protocol ha...
Source: Revista Argentina de Microbiologia - Category: Microbiology Tags: Rev Argent Microbiol Source Type: research