Adaptation of an Invader ® assay for the detection of African swine fever virus DNA

Abstract

A closed tube isothermal Invader ® assay (Third Wave Technologies Inc., Madison, Wisconsin, USA) was adapted for the detection of African swine fever virus (ASFV) DNA. Several ASFV Invader ® assays were designed successfully and tested on a real-time PCR instrument (iCycler™, BioRad). The assay exhibiting the lowest signal/noise ratio (VP73 ASFV Invader ® Assay) was analysed further using serial 10-fold dilutions of Lisbon 60 ASFV viral genome. The assay sensitivity was determined to be in the order of 2500 copies of ASFV DNA and showed a dynamic range of 4 logs, from 2.5 × 10 6 to 2500 copies. The high specificity of the test was demonstrated by the lack of cross-reactivity to the clinically similar but heterologous virus, classical swine fever virus. The sensitivity of the Invader ® assay is sufficient for the testing of acutely infected viremic animals in which the viral load will be high. The robustness and ease of use of the ASFV Invader ® assay, combined with the possibility to run and read the assay using simple and relatively inexpensive equipment, makes it suitable for laboratories lacking containment facilities and/or real-time PCR instrumentation or on a regional basis for on-site diagnosis close to putative sites of ASFV outbreaks.