title: Detection of viable enterotoxin-producing Bacillus cereus and analysis of toxigenicity from ready-to-eat foods and infant formula milk powder by multiplex PCR.
abstract: Bacillus cereus is responsible for several outbreaks of foodborne diseases due to its emetic toxin and enterotoxin. Enterotoxins, cytotoxin K (CytK), nonhemolytic enterotoxin (Nhe), and hemolysin BL (Hbl), have been recorded in several diarrheal cases due to food poisoning from B. cereus. The objective of this study was to develop a rapid and accurate method that combines multiplex PCR with propidium monoazide to selectively detect viable cells of enterotoxin-producing B. cereus in milk powder, noodles, and rice, and investigate the distribution of enterotoxins in 62 strains of B. cereus in Jiangxi province, China. The specificity of primers of 3 enterotoxins (i.e., cytK, nheA, and hblD) of B. cereus was verified by inclusivity and exclusivity tests using single PCR. Upon optimization of multiplex PCR conditions, it was found that the detection limit of viable cells was 10(2) cfu/mL of B. cereus in pure culture. By enrichment for 3 or 4 h and propidium monoazide pretreatment, a protocol for detection of viable cells as low as 2.2×10(1) cfu/g in spiked food (e.g., milk powder, noodles, and rice) was established and proved valid even under the interference of non-Bacillus cereus at as high as 10(5) cfu/g. Moreover, the protocol based on multiplex PCR for detection was applied for the analysis of distribution of toxin gene of B. cereus, and the results showed a regional feature for toxin gene distribution, indicating that potential toxigenicity of B. cereus should be evaluated further.
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Hengyi
authors.initials: H
authors.lastname: Xu
authors.affiliation: Institute of Nutrition and Food Safety, Jiangxi Provincial Centre for Disease Control, Nanchang 330046, P. R. China.
authors.forename: Chengwei
authors.initials: C
authors.lastname: Liu
authors.affiliation: Food and Nutritional Science, School of Biological Science, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
authors.forename: Nagendra P
authors.initials: NP
authors.lastname: Shah
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China. Electronic address: weihua114@live.cn.
authors.forename: Hua
authors.initials: H
authors.lastname: Wei
mesh_terms: D000818:Animals; D001409:Bacillus cereus / Q000235:genetics / Q000302:isolation & purification*; D004768:Enterotoxins / Q000737:chemistry / Q000302:isolation & purification*; D057140:Fast Foods / Q000382:microbiology*; D005516:Food Microbiology; D006801:Humans; D007223:Infant; D041943:Infant Formula / Q000382:microbiology*; D008892:Milk / Q000737:chemistry / Q000382:microbiology; D060885:Multiplex Polymerase Chain Reaction
chemical_list: D004768:Enterotoxins
keywords: Bacillus cereus; enterotoxin-producing; multiplex PCR; propidium monoazide; toxigenicity