Dataset Information

A Visualized Isothermal Amplification Method for Rapid and Specific Detection of Emetic and Non-emetic Bacillus cereus in Dairy Products.

ABSTRACT: Bacillus cereus is widely distributed in foods, especially dairy products, and can lead to diarrhea (non-emetic B. cereus) and emesis (emetic B. cereus). Although diarrhea due to B. cereus is usually mild, emesis can lead to acute encephalopathy and even death. To develop rapid and sensitive detection methods for B. cereus in foods, specific primers targeting the gyrase B (gyrB) and cereulide synthetase (ces) genes were designed and screened using recombinase polymerase amplification (RPA). Probes and base substitutions were introduced to improve specificity and eliminate primer-dependent artifacts. The 5' ends of the reverse primers and probes were modified with biotin and fluorescein isothiocyanate for detection of RPA products on a lateral flow strip (LFS). The developed RPA-LFS assay allows detection within 20 min at 37°C with no cross-reactivity with other foodborne pathogens. The limit of detection was 10⁴ copies/ml and 10² CFU/ml in pure cultures and milk, respectively. Comparisons with established methods using cream obtained similar results. A specific, rapid, and sensitive RPA-LFS assay was successfully developed for on-site detection of B. cereus in dairy products to distinguish emetic from non-emetic strains.

SUBMITTER: Wang L

PROVIDER: S-EPMC8996228 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

A Visualized Isothermal Amplification Method for Rapid and Specific Detection of Emetic and Non-emetic Bacillus cereus in Dairy Products.

Wang Lei L Yang Huansen H Wang Kun K Yang Haitao H Zhao Mengdi M Shang Yuping Y Wang Fang F Dong Jingquan J Zhao Weiguo W Li Li L Liang Wei W Wang Yan Y

Frontiers in microbiology 20220328

Bacillus cereus is widely distributed in foods, especially dairy products, and can lead to diarrhea (non-emetic B. cereus) and emesis (emetic B. cereus). Although diarrhea due to B. cereus is usually mild, emesis can lead to acute encephalopathy and even death. To develop rapid and sensitive detection methods for B. cereus in foods, specific primers targeting the gyrase B (gyrB) and cereulide synthetase (ces) genes were designed and ...[more]

PMID: 35418965

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Project description:Vibrio cholerae is a leading waterborne pathogenic bacterium worldwide. It can cause human cholera that is still pandemic in developing nations. Detection of V. cholerae contamination in drinking water and aquatic products is imperative for assuring food safety. In this study, a simple, sensitive, specific, and visualized method was developed based on loop-mediated isothermal amplification (LAMP) (designated sssvLAMP) to detect virulence-associated (ctxA, tcpA, hapA, mshA, pilA, and tlh) and species-specific (lolB) genes of V. cholerae. Three pairs of oligonucleotide primers (inner, outer, and loop primers) were designed and or synthesized to target each of these genes. The optimal conditions of the sssvLAMP method was determined, and one-step sssvLAMP reaction was performed at 65°C for 40 min. Positive results were simply read by the naked eye via color change (from orange to light green) under the visible light, or by the production of green fluorescence under the UV light (260 nm). The sssvLAMP method was more efficient in detecting 6.50 × 101-6.45 × 104-fold low number of V. cholerae cells, and more sensitive in V. cholerae genomic DNA (1.36 × 10-2-4.42 × 10-6 ng/reaction) than polymerase chain reaction (PCR) method. Among 52 strains of V. cholerae and 50 strains of non-target species (e.g., other Vibrios and common pathogens) examined, the sensitivity and specificity of the sssvLAMP method were 100% for all the target genes. Similar high efficiency of the method was observed when tested with spiked samples of water and aquatic products, as well as human stool specimens. Water from various sources and commonly consumed fish samples were promptly screened by this simple and efficient visualized method and diversified variation in the occurrence of the target genes was observed. V. cholerae strains could be mostly detected by the presence of hapA and tlh alone or in combination with other genes, indicating a variable risk of potentially pathogenic non-O1/O139 strains in edible food products. This novel LAMP method can be a promising tool to address the increasing need of food safety control of aquatic products.

Dataset Information

A Visualized Isothermal Amplification Method for Rapid and Specific Detection of Emetic and Non-emetic Bacillus cereus in Dairy Products.

Publications

A Visualized Isothermal Amplification Method for Rapid and Specific Detection of Emetic and Non-emetic <i>Bacillus cereus</i> in Dairy Products.

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