Development of a novel high resolution melting assay for identification and differentiation of all known 19 serovars of Actinobacillus pleuropneumoniae

In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinicalA. pleuropneumoniae isolates. The novel HRM comprises the species-specific APP-HRM1 and two serovar-specific HRM assays (APP-HRM2 and APP-HRM3). APP-HRM1 allowed polymerase chain reaction (PCR) amplification ofapxIV resulting in anA. pleuropneumoniae specific melting curve, whilenadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user-friendly assay showed high sensitivity with 1.25  fg–125 pg of input DNA and a specificity of 100% to identifyA. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen.
Source: MicrobiologyOpen - Category: Microbiology Authors: Tags: ORIGINAL ARTICLE Source Type: research