Multiplex-PCR as an identity assay for Mycobacterium bovis BCG Moreau descendants

In the study, we assessed the identity of locally produced BCG vaccine via screening for the presence of genetic markers specific for particular Mycobacterium bovis BCG substrains - RD8, RD2, senX3-regX3, RD14, RD16, ΔRD1, DU2, a second copy of IS6110, mutation D322G in phoR, and deletions in fadD26-ppsA and Rv3887c regions. In order to increase the specificity of the multiplex-PCR test for locally produced BCG vaccine, we have modified previously developed primer sets by the introduction of a primer pair specific for deletion in Rv3887c. The modified multiplex-PCR specifically and reproducibly distinguished both BCG Moreau sublineages, and allowed, with no decrease in power, differentiation of BCG substrains of different origin. The growing knowledge of genetic differences among BCG vaccine strains enables improvements in the specificity of identity tests that will be useful both for routine release of vaccines and potential applications in clinical practice. Modified multiplex-PCR accompanied by PFGE analysis can serve as specific tools to monitor consistency in BCG manufacture.