Figure 2. Genetic characterization of the BCG.HIVA^CAT.

GenoType MTBC assay and Multiplex PCR assay. (A) The BCG.HIVA^CAT strain identification results representative of all of the patterns obtained with the GenoType MTBC assay. The positions of the oligonucleotides, the marker line and the BCG hybridization pattern are shown on the right. The specificity and targeted genes of the lines are as follows: 1, conjugate control; 2, amplification control (23S rRNA); 3, MTBC specific (23S rRNA); 4 to 12, discriminative for the MTBC species (gyrB); 13, M. bovis BCG (RD1). The samples analyzed were: Strip 1: BCG Connaught; Strip 2: BCG.HIVA^CAT; Strip 3: BCG.HIVA²²²; Strip 4: BCG wild type. All four strains presented the same hybridization pattern corresponding to M. bovis BCG. (B) The BCG.HIVA^CAT Pasteur substrain identification by multiplex PCR assay. Lane 1 and 12: molecular weight marker; lane 2: negative control; lane 3, 6–11: BCG.HIVA^CAT(clone10); lane 4: BCG Danish strain (using 1 µl of template); lane 5: BCG Danish strain (using 4 µl template); The samples were analyzed by multiplex primer assay or single primer pair assay. Lane 2–5: multiplex primers; lane 6: ET1-3 primers; lane 7: RD2 primers; lane 8: RD8 primers; lane 9: RD14 primers; lane 10: RD16 primers; lane 11: C3–C5 primers. (C) Enzymatic restriction analysis of pJH222.HIVA^CAT plasmid DNA extracted from both the Master Seed (MS, lanes 1–5) and the Working Stock (WS, lanes 7–11) of BCG.HIVA^CAT cultures. Lane 1 and lane 7: uncut plasmid; lane 2 and lane 8: HpaI digestion; lane 3 and 9: KpnI digestion; lane 4 and 10: digestion with SpeI; lane 5 and 11: digestion with HindIII; lane 6 and 12: Molecular Weight Marker (1 kb Plus, Invitrogen). (D) PCR analysis of HIVA DNA coding sequence using as template the cultures of BCG.HIVA^CAT Master Seed (lane 1), and Working Stock (lane 2), Molecular Weight Marker (lane 3), positive control plasmid DNA pJH222.HIVA (lane 4).