MOL-PCR and xMAP technology – a novel approach to the detection of African swine fever virus DNA

African swine fever (ASF) is highly contagious haemorrhagic viral disease of domestic pigs and wild boars. The causative agent can be transmitted by direct contact with infected animals or via a contaminated environment, fomites, feed, meat and products thereof. Soft ticks (genus Ornithodoros) are known reservoirs and transmission vectors of the virus. As the disease causes serious problems in many countries, rapid detection of the agent and early diagnosis could help in prevention of its spread. Therefore, a multiple-analyte profile (xMAP) technology based on multiple oligonucleotide ligation followed by polymerase chain reaction (MOL-PCR) was introduced and verified. A system targeting two independent loci of the virus genome was designed to increase the likelihood of different strains detection and an internal control was employed to verify the correct course of the analysis. The sensitivity was experimentally determined as 10 genomic copies of the virus in one µl of isolated DNA. The system was verified on samples originating from a recent ASF outbreak in the Czech Republic (six spleen) and the Czech market (eight liver and heart tissues) with real-time polymerase chain reaction used as a reference method. The results of both methods were in agreement, even in samples with a low concentration of the virus genome (9.45 × 101 genomic copies/µl of DNA). The system introduced represents an open method allowing the detection and semi-quantification of up to 50 targets/agents in one reaction. It can, therefore, be used for rapid one-step screening and as an effective tool for risk management.