<p>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].</p>
<p><em>Bacillus cereus</em>(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]. <em>B.cereus</em>, 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, <em>B. cereus</em> 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].</p>
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<h2><strong><em> Current Problem</strong></em></h2>
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<p> Although abundant types of methods of detecting B.cereus have been developed, most of them are time-consuming and rely heavily on lab equipment. </p>
<p> Although abundant types of methods of detecting <em>B.cereus</em> have been developed, most of them are time-consuming and rely heavily on lab equipment. </p>
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<h4><strong><em>1. Traditional methods</strong></em></h4>
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<p>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 B. 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 B. cereus agar plates since the metabolic activity of B. 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.</p>
<p>The accuracy of the detection of <em>Bacillus cereus</em> is hindered by the similarity of other strains in the <em>Bacillus cereus</em> group to <em>Bacillus cereus</em> [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.</p>
<p>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 B. 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 B. thuringiensis and B.cereus due to the extreme genetic similarity between them. Moreover, this method will take longer time than the normal PCR. [11].</p>
<p>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].</p>
<p>Immunological assay against B. 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 B. 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 B. 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.</p>
<p>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.</p>
<p>These assays, including a WST-1-based assay and an MTT-based assay, rely on the cytotoxicity of B. cereus toxin on mammalian cells. Both assays involve adding B. 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]. </p>
<p>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]. </p>
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<h2><strong><em>An overview of designs</strong></em></h2>
