Bacillus cereus(B.C.), a type of gram-positive, pathogenic bacteria, is one of the most common causes of foodborne diseases. Although it mostly causes vomiting and diarrhea when found in root crops and meat/dairy products, respectively, it could also cause severe respiratory disease or even trigger acute liver failure and leads to death in some cases [1]. B.cereus, especially its spores, are able to resist high temperatures. Thus they cannot be destroyed by normal cooking and heating processes [1]. It is very unnerving to find out that food around us is carrying a potentially deadly pathogen. Besides food poisoning, B. cereus induces local and systemic infections. The main described conditions are septicemia, endophthalmitis, pneumonia, endocarditis, meningitis, and encephalitis, especially in immunosuppressed individuals such as neonates, resulting in patient death in about 10% of cases. In addition, several cases of fulminant infections similar to anthrax, and affecting healthy persons, have also been reported [2].
Although abundant types of methods of detecting B.cereus have been developed, most of them are time-consuming and rely heavily on lab equipment.
The accuracy of the detection of Bacillus cereus is hindered by the similarity of other strains in the Bacillus cereus group to Bacillus cereus [3]. The traditional method is to extract the Bacillus cereus from food samples and culture them on agar plates. They are then detected morphologically and biochemically [4]. The identification of colonies can be aided by chromogenic Bacillus cereus agar plates since the metabolic activity of Bacillus cereus changes the color of the medium [5]. However, small and medium-sized food workshops usually cannot provide materials, instruments, and specialized technicians to perform such time-consuming identification.
PCR has also become a method to detect Bacillus cereus strains [6], due to many toxin-related genes, such as bceT (encoding enterotoxin T) [6], hblC, hblD, hblA, hblB (encoding components of hemolysin BL) [7], ces (encoding mevalonate) [8], and nheA, nheB, nheC (encoding components of hemolytic enterotoxins) [9] were identified. However, PCR targeting a single toxin-related gene is not sufficient to exclude all possible toxins because different strains in the Bacillus cereus group have different types of toxin genes [10]. Advanced amplification and electrophoresis methods such as rep-PCR, RAPD-PCR, and PCR-TTGE may be able to serve as a solution. But this method still cannot distinguish Bacillus thuringiensis and Bacillus cereus due to the extreme genetic similarity between them. Moreover, this method will take longer time than the normal PCR. [11].
Immunological assay against Bacillus cereus toxin is an accurate method to detect toxic strains. Mouse monoclonal antibodies and rabbit antisera can be used against each of the three components of hemolysin BL (HBL) and each of the three components of non-hemolytic enterotoxin (Nhe), both diarrhea-related three-component enterotoxins from B. cereus that have been well developed and characterized [12] [13]. In addition, rabbit antisera and mouse monoclonal antibodies against the N-terminal end of the Bacillus cereus flagellin are recently being developed and are proving to be powerful tools for the detection of B. cereus [14]. However, the catalytic toxin Cerulidein of Bacillus cereus cannot be detected by immunological methods, as it is not antigenic [15]. Also, the cost of antibodies and detection equipment is unaffordable for developing regions.
These assays, including a WST-1-based assay and an MTT-based assay, rely on the cytotoxicity of Bacillus cereus toxin on mammalian cells. Both assays involve adding Bacillus cereus culture supernatants to a Chinese hamster ovary (CHO) cell line culture (other mammalian cell lines may also be acceptable) and measuring the metabolites of the cells. More specifically, reductase in the mitochondria of living cells can reduce WST-1 to soluble formazan [17] or MTT to insoluble formazan [16], but cells killed by B. cereus toxin cannot. A functional relationship can be established by cytotoxicity, the percentage of viable cells and the absorbance of formazan. However, the MTT assay takes at least 44-52 hours to complete, whereas the WST-1 assay takes only 3 hours to complete because of the longer lysis process of Formazan [17].
To solve the problem, we plan to make a convenient, cheap, and broadly applicable detection kit. The kit uses endolysin to lys B.cereus cells and detect the ATP or DNA it released.
Endolysin have two specific domains (enzymatically active domain, EAD, and cell-binding domain, CBD), in which they recognize and bind with specific cell wall structures and lyse the cell. They are well-studied and easy to be applicated, and they can be simply folded and expressed in E.coli, which enables massive production. We plan to find the CBD with the highest binding specificity and the EAD with the highest cleavage specificity, fuse them together, and express them in E.coli.
In terms of detecting DNA, we choose the one-HOur Low-cost Multipurpose highly Efficient System (HOLMES), a technique that can detect specific sequences and release optical signals even if the templates are low in concentration. It depends on the trans activity of Cas12B and is very accurate.
ATP detection is based on the reaction of luciferin and luciferase with the presence of ATP, which release detectable fluorescence. Although not as accurate as HOLMES, it enables the user to use an App to read the fluorescence intensity on phones conveniently.
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