Volume 69 (2019) Issue: 2019 No#4

Duplex PCR for detection of Aleutian disease virus from biological and environmental samples

Author(s): Kowalczyk Marek, Jakubczak Andrzej, Gryzińska Magdalena

Keywords:Aleutian disease, diagnostics, duplex PCR, optimization

Aleutian disease is one of the most serious disease entities affecting mink farms. The disease causes significant economic losses in mink breeding countries. The aim of the study was to optimize a diagnostic test based on duplex PCR to enable detection of Aleutian disease virus in biological and environmental samples. Blood (n = 40) and spleen (n = 40) samples from animals with suspected infection, and swabs from cages in which infected animals were kept (n = 20) were used for analysis. DNA was isolated from the samples, followed by optimization of the duplex PCR reaction targeting sequences coding NS1 and VP2 proteins. The qPCR method was used to determine the sensitivity of the reaction. The specificity of the analysis was confirmed by the sequencing results. Optimized duplex PCR enabled detection of Aleutian Mink Disease Virus (AMDV) genetic material in biological and environmental samples. Testing of the sensitivity of the method indicated clear amplification for both primer pairs at 102 copies of viral DNA in a reaction. Sequencing confirmed the specificity of the reaction, which in the case of both primer pairs indicated an over 90% agreement between the isolates and the variants of the virus from the databases. The use of duplex PCR to detect two regions of the AMDV genome may increase the sensitivity and specificity of the method and significantly expand the possibilities of further analysis based on sequencing.