The efficiency of a novel biomarker (the transcriptional regulator,
XRE
) was tested and evaluated in differentiating
Bacillus thuringiensis
from
Bacillus cereus
group species in environmental and spiked samples based on PCR and real-time PCR. Totally 120 strains, representing two bacterial groups,
B. cereus
group and non-
Bacillus
sp., were used to evaluate the performance of
XRE
and crystal protein (
cry
2, an existing biomarker). Further, three diverse samples (kimbap, lettuce, and spinach) were inoculated with
B. thuringiensis
and prominent biomarkers
XRE
and
cry
2 were used as targets. Direct analysis of the detection results for the pure cultures of
B. cereus
group wild-types, references and type strains revealed an accuracy rate of 97.5% targeting
XRE
, and 83.3% targeting
cry
2. The real-time PCR was constructed with a
R
2
-value of 0.993. For the artificially contaminated samples, a concentration of 10
3
CFU/g of
B. thuringiensis
in spiked food samples could be detected using real-time PCR targeting
XRE.
A good performance was obtained with
XRE
in discriminating
B. thuringiensis
from
B. cereus
groups, as well as detecting
B. thuringiensis
in spiked food samples with PCR or real-time PCR. Therefore, this real-time PCR targeting X
RE
can be used as a dependable and promising tool to identify
B. thuringiensis
in foods.