Proposal and requirements for preparing panC sequence
Partial amplification of panC gene and sequencing:
PCR amplification can be performed on cellular suspension or DNA issue from
different preparation methods : frozen/defrosted bacterial cells
and then heated up to 90°C during 10 min ; semi-purified DNA prepared with ‘Instagene Matrix” kit (Biorad) ; purified DNA
(example of a rapid method in Guinebretičre et al., 2003).
The final PCR mixture (90 µl) may contain 300 ng of DNA template, 0.2 mM
dNTP mix (Eurogentec, Seraing, Belgium), 2.5 mM MgCl2, 0.25 µM
Thermal cycling can be carried out in a thermocycler with the following run:
a starting cycle of 5 min at 94°C, followed by 30 cycles of 15 sec at 94°C, 30 sec at 55°C,
of each primer, 0.75 U of AmpliTaq polymerase (Perkin-Elmer, Courtaboeuf, France) and 9 µl of 10X AmpliTaq buffer without MgCl2
(Perkin-Elmer, Courtaboeuf, France). For all groups except group VII, the following primers can be used : 5’- TYGGTTTTGTYCCAACRATGG-3’
(Forward degenerated primer) and 5’-CATAATCTACAGTGCCTTTCG-3’ (Reverse). For group VII strains, reverse primer 5’- CATAATCAACTATACCGTTTG-3’
can be substituted.
and 30 sec at 72°C, and a final extension of 7 min at 72°C.
PCR products can be purified using « High Pure PCR Product » kit (Roche Diagnostics,
Mannheim, Germany) and sent for sequencing, using primer
5’-ATAATCTACAGTGCCTTTCG-3’for all strains except group 7 strains and primer 5’-ATAATCAACTATACCGTTTG-3’ for group 7 strains.
As group VII strains are relatively rare, a strategy consists to use first primers recommended for all groups except group VII, and
if the reaction is negative then use primers recommended for group VII.
Preparation of sequence:
-The sequence should be in fasta format (reverse complemented if necessary) and at least 250 bases length.
-A, C, G, T are the only characters to be permitted.
-If no identification is obtained, presence of the initial sequence 5’-AACAAAC-3’ and /or 5’-AACAGAC-3’
may be checked.This sequence must be
present on your sequence(s) to initiate alignments (position 305-311 on panC sequence recorded for ATCC 14579). Absence of this sequence
would signify that your sequence does not belong to the B. cereus Group or that error occured during sequencing.
Guinebretière M.-H., Nguyen-The C. Sources of Bacillus cereus contamination
in a pasteurized zucchini purée processing line, differentiated by two
PCR-based methods. FEMS Microbiol. Ecol. (2003) 43 :207-215.