Recombinant vaccinia viruses are popular tools for the delivery of heterologous genes for
vaccination purposes. Vaccinia viruses are generally classified as risk group 2 biological
agents (in accordance to directive 2000/54/EC), with the attenuated strain Modified
vaccinia virus Ankara (MVA) being classified into risk group 1. For the analytical
surveillance of genetic engineering operations with vaccinia viruses, developing methods
that enable the differentiation between risk group 2 vaccinia strains and MVA and that
allow the identification of possibly inserted heterologous genes and their integration
loci is important. This article describes a method based on multiplex ligation-dependent
probe amplification (MLPA), which has been customized to simultaneously check 13 different
vaccinia viral genome sequences. The presence or absence of MLPA products points towards
the utilized vaccinia viral backbone and the integration loci of heterologous genes.
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