Development of PCR based molecular diagnosis kit for rapid detection of animal diseases: A model study

B. anthracis is the causative agent of anthrax, which is primarily affecting herbivores but can be seen in all mammalians. Pathogenicity of B. anthracis is based on the virulence genes carried on two plasmids, namely pXO1 and pXO2. While pXO1contains anthrax toxin genes, pXO2 plasmid carries the gene operon responsible for the synthesis of a capsule which enables the bacteria to evade the immune system of the host organism. B. anthracis is a member of the B. cereus group and shows a highly monomorphic lineage. Many B. anthracis polymerase chain reaction (PCR) based diagnosis kits developed up to now rely on the detection of marker genes carried on the virulence plasmids, relatively few assays incorporate chromosomal DNA markers due to high similarity of the B. anthracis to other B. cereus group members at the chromosomal DNA level. However, in recent years, B cereus strains that carry pXO1 plasmid were discovered and strains with both pXO1 and pXO2 have been isolated from African great apes. Besides there are B. thuringiensis strains harboring pXO2 capsule genes. Therefore, presence of pXO1 and pXO2 is insufficient in discriminating B. anthracis from other Bacilli. In this study, two different multiplex PCR kits targeting B. anthracis- specific chromosomal lambdoid prophage region besides the virulence genes on the pXO1 and pXO2 plasmids were developed. One of these kits is completely specific to this study and provides the detection of the chromosomal region with a dual-stage binding probe, which can be used for the environmental samples as successfully as pure DNA samples. As for the second kit, it is a diagnosis kit that can be used alternatively and formed by adapting primer and probe sets existing in the literature. Furthermore, internal control fragments were designed in order to evaluate false-negative results. With this study, optimization of the two kits presented here was performed and their ability to work successfully in a quadruple multiplex setting was shown. At the same time, exclusivity of the kits were tested by using a panel of Bacillus species genetically very close to B. anthracis.