Primers used for MLST following the Tourasse-Helgason scheme

The Bacillus cereus group MLST scheme developed by Tourasse, Helgason et al. 2006 is based on internal fragments of the following seven house-keeping genes: 

PCR Amplification

Primers used for PCR amplification and sequencing of B. cereus group housekeeping genes:

Gene

Fragment length (bp)§

Forward primer 5'-3' sequence

Reverse primer 5'-3' sequence

Annealing temp. °C

adk*

411 (648)

CAG CTA TGA AGG CTG AAA CTG

CTA AGC CTC CGA TGA GAA CA

57

glpT*

330 (1,347)

TGC GGC TGG ATG AGT GA

AAG TAA GAG CAA GGA AGA

59

glpF

372 (822)

GCG TTT GTG CTG GTG TAA G

CTG CAA TCG GAA GGA AGA AG

52

panC#

350 (849)

CGA TAT CCT CGT GAT ATT GAT AGA G

TCC GCA TAA TCT ACA GTG CCT TTC

57

pycA

363 (3,447)

GCG TTA GGT GGA AAC GAA AG

CGC GTC CAA GTT TAT GGA AT

57

ccpA*

418 (996)

GTT TAG GAT ACC GCC CAA ATG

TGT AAC TTC TTC GCG CTT CC

59

pta

414 (972)

GCA GAG CGT TTA GCA AAA GAA

TGC AAT GCG AGT TGC TTC TA

58

§the numbers in parentheses are total lengths of the genes.
*primers for adk, glpT, and ccpA are from Helgason et al., 2004
primers for glpF, pycA, and pta are from Priest et al., 2004
#primers for panC are from Candelon et al., 2004 and Sorokin et al., 2006

NEW: it is now also possible to use an isothermal set of primers, with a common annealing temperature of 55 °C. These primers were designed by Dr. Alexei Sorokin, INRA, France.

Gene

Fragment length (bp)§

Forward primer 5'-3' sequence

Reverse primer 5'-3' sequence

adk*

411 (648)

GGT AAA GGT ACA CAA GCC GAA CAG

CTA AGC CTC CGA TGA GAA CAT CG

glpT*

330 (1,347)

ATG GGC TGG TAT TCC AGG TAC AC

AAG TAA GAG CAA GGA AGA ACA TTG CA

glpF

372 (822)

TTG TTA ATC GTA CTT GGT GGC GG

ACT GGA ATC CAT GCA TAC TTC CAG TT

panC#

350 (849)

CGA TAT CCT CGT GAT ATT GAT AGA G

TCC GCA TAA TCT ACA GTG CCT TTC

pycA

363 (3,447)

CTA TGC GTT AGG TGG AAA CGA AAG AC

GTC GGT GTA TCA AGC ACA GAT ACA T

ccpA*

418 (996)

TAT GAT GTA GCG CGT GAA GC

CCT TGT AAC TTC TTC GCG CTT CC

pta

414 (972)

CGC TGC AGA GCG TTT AGC AAA AGA A

CTC AGC TAC AGA TGG TAC GAA TGC

§the numbers in parentheses are total lengths of the genes.

 

PCR protocol

2μl of each primer (20μM)

5μl 2mM dNTP

5μl buffer

1μl polymerase enzyme

1-2μl template from lysed bacteria extract or 50nmol DNA

Xμl MQ water. Total of 50μl


Cycle program:

1         4 min @ 94°C

2         1 min @ 94°C

3         1 min @ annealing temp of primer (see table above)

4         1 min @ 72°C

5         repeat from step 2 39 times (a total of 40 cycles)

6         7 min @ 72°C

7         ∞ min @ 4°C


Same sets of primers are used for sequencing protocol.

 

Whichever set of primers you use (isothermal or not), only that set is needed for amplifying each gene. PCR amplification can be done using fresh boiled cells as a template. For details see Helgason et al. 2004

Same primer pairs for sequencing as used for PCR amplification.

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