Differentiation of Bacillus thuringiensis From Bacilluscereus Group Using a Unique Marker Based on Real-Time PCR

Abstract

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.

Study on the Identification Methods for Effective Microorganisms in Commercially Available Organic Agriculture Materials

“…Furthermore, the primer was tested on the isolates from AMEM, and the results demonstrated the amplification of a ~246 bp fragment in isolate D-1 and confirmed it as B. thuringiensis ( Figure 5 c). The results are consistent with the findings of Wei et al (2019) [ 16 ], showing the high specificity of XRE primers for identifying B. thuringiensis . Moreover, it has been well-documented that B. thuringiensis selectively produces insecticidal crystal proteins [ 16 , 23 ], which can be utilized as an appropriate marker for proper identification.…”

“…The results are consistent with the findings of Wei et al (2019) [ 16 ], showing the high specificity of XRE primers for identifying B. thuringiensis . Moreover, it has been well-documented that B. thuringiensis selectively produces insecticidal crystal proteins [ 16 , 23 ], which can be utilized as an appropriate marker for proper identification. Herein, we examined the presence of crystal proteins using crystal protein staining and observed the presence of these crystal proteins in isolate D-1 ( Figure 6 ).…”

“…In order to identify B. subtilis , B. thuringiensis , and B. licheniformis at the species level, species-specific PCR was performed. The species-specific primers were designed for the ytcP gene of B. subtilis (F 5′-CTTACGGGTTATCCCGC-3′ and R 5′-CCGACCCCATTTCAGACATATC-3′) [ 15 ], XRE gene of B. thuringiensis (F 5′-AAGATATTGCAAGCGGTAAGAT-3′ and R 5′-GTTTTGTTTCAGCATTCCAGTAC-3′) [ 16 ], and Blich gene of B. licheniformis (F 5′-AKACGGAAGTGACGGGAAC-3′ and R 5′-AGAAACTTTTCRAGCGCTT-3′) [ 12 ]. The total volume of the reaction mixture for PCR was 25 μL, which contained DNA template (1.0 μL, 50 ng), GoTaq ® G2 Green Master Mix (12.5 μL, pH 8.3, 1.5 mM MgCl 2 , 200 μM of each dNTP, DNA polymerase), species-specific forward primer (1.0 μL), reverse primer (1.0 μL), and nuclease-free distilled water (9.5 μL).…”

Bacillus Thuringiensis – Nowy PotencjaŁ Aplikacyjny

“…Gatunek B. thuringiensis należy do domeny Bacteria, typu Firmicutes, klasy Bacilli, rzędu Bacillales, rodziny Bacillaceae i rodzaju Bacillus [67]. Rodzaj Bacillus dzieli się na trzy grupy, przy czym B. thuringiensis jest zaliczany do grupy B. cereus, do której należą B. anthracis, B. cereus, B. cytotoxicus, B. mycoides, B. pseudomycoides, B. toyonensis, oraz B. weihenstephanensis [86]. Przez ostatnie 100 lat, ze względu na różnice w morfologii ich kolonii, właściwości metaboliczne, w tym oporność na penicylinę, bakterie te opisywano jako odrębne gatunki [58].…”

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“…The nearby strains were used for maximum likelihood phylogenetic analyses. However, the use of other markers such as gyrB and XRE is recommended (Helgason et al, 2000;Punina et al, 2013;Wei et al, 2019) to identify all strains up to the species taxonomic level (Helgason et al, 2000;Punina et al, 2013;Wei et al, 2019).…”