Development of a novel high resolution melting assay for identification
and differentiation of all known 19 serovars of Actinobacillus
pleuropneumoniae
Abstract
Actinobacillus pleuropneumoniae is the etiological agent of porcine
pleuropneumonia, a respiratory infectious disease responsible for global
economic losses in the pig industry. From a monitoring perspective as
well as due to the different courses of disease associated with the
various serovars, it is essential to distinguish them in different herds
or countries. 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 clinical A. 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
PCR amplification of apxIV resulting in an A. pleuropneumoniae specific
melting curve, while nadV specific primers differentiated biovar 2 from
biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13 A. 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 identify A. 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.