Development of real-time PCR method based on different target genes for rapid detection of Bacillus cereus
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摘要: 为了建立一种基于不同靶基因的快速灵敏检测食品中产与不产呕吐毒素蜡样芽孢杆菌的荧光定量PCR方法,本研究采用煮沸法提取基因组DNA,用普通PCR方法验证引物的特异性,通过在米饭中添加不同浓度的产呕吐毒素蜡样芽孢杆菌模拟受污染的食品,采用本研究构建的荧光定量PCR方法对米饭进行检测。结果表明基于不同靶基因设计的两对引物具有特异性强和扩增效率高等优点,荧光定量PCR方法能对污染米饭中蜡样芽孢杆菌准确定量,检测限能达到101CFU/g。建立的荧光定量PCR方法特异、灵敏和准确,适用于食品中蜡样芽孢杆菌的快速定量检测。Abstract: To develop a rapid and sensitive real-time PCR method to differentiate emetic Bacillus cereus and non- emetic B.cereus based on different target genes.DNA was isolated by boiling method and used to test the specificity of primers by PCR.The rice was inoculated by emetic B.cereus for imitating contaminated food and detected by real- time PCR method.The results showed that primers were specific and high of amplification efficiency, the B.cereus could be quantified accurately by real-time PCR and the limit of detection was 101CFU /g in the contaminated rice.The novel developed real- time PCR method was specific, sensitive and accurate, which could be used for the rapid detection of B.cereus in food.
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Keywords:
- Bacillus cereus /
- cesB /
- gyrB /
- real-time PCR /
- detection
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[1] Rosenbaum A, Papaliodis D, Alley R M, et al.Bacillus cereus Fasciitis:A unique pathogen and clinically challenging sequela of inoculation[J].American Journal of Orthopedics, 2013, 42 (1) :37-39.
[2] Granum P E, Lund T.Bacillus cereus and its food poisoning toxins[J].FEMS Microbiology Letters, 2006, 157 (2) :223-228.
[3] 仪淑敏, 李远钊, 张培正, 等.蜡样芽孢杆菌在营养肉汤和维也纳香肠中的生长模型及控制[J].食品工业科技, 2007, 28 (9) :72-75. [4] Ehling-Schulz M, Fricker M, Grallert H, et al.Cereulide synthetase gene cluster from emetic Bacillus cereus:structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1[J].BMC Microbiology, 2006, 6 (1) :1-11.
[5] Ehling-Schulz M, Fricker M, Scherer S.Bacillus cereus, the causative agent of an emetic type of food-borne illness[J].Molecular Nutrition&Food Research, 2004, 48 (7) :479-487.
[6] 刘志勇, 赵雪涛, 陆敏.Real-time PCR测定食品中蜡样芽胞杆菌的数量[J].中国卫生检验杂志, 2012, 22 (8) :1858-1863. [7] Fricker M, Messelhuβer U, Busch U, et al.Diagnostic realtime PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks[J].Applied and Environmental Microbiology, 2007, 73 (6) :1892-1898.
[8] Shiota M, Saitou K, Mizumoto H, et al.Rapid detoxification of cereulide in Bacillus cereus food poisoning[J].Pediatrics, 2010, 125 (4) :951-955.
[9] Dzieciol M, Fricker M, Wagner M, et al.A novel diagnostic real-time PCR assay for quantification and differentiation of emetic and non-emetic Bacillus cereus[J].Food Control, 2012, 32 (2013) :176-185.
[10] 刘仲敏, 郑鸣, 王永芬, 等.食源性沙门氏菌实时定量PCR检测研究[J].食品工业科技, 2007, 28 (8) :233-236. [11] Yamada S, Ohashi E, Agata N, et al.Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B.thuringiensis, B.mycoides, and B.anthracis and their application to the detection of B.cereus in rice[J].Applied and Environmental Microbiology, 1999, 65 (4) :1483-1490.
[12] Ombui J N, Gitahi J N, Gicheru M M.Direct detection of Bacillus cereus enterotoxin genes in food by multiplex polymerase chain reaction[J].International Journal of Integrative Biology, 2008, 2 (3) :172-181.
[13] Lim J S, Kim M R, Kim W, et al.Detection and differentiation of non-emetic and emetic Bacillus cereus strains in food by real-time PCR[J].Journal of the Korean Society for Applied Biological Chemistry, 2011, 54 (1) :105-111.
[14] Beattie S H, Williams A G.Detection of toxins[M].Encyclopedia of Food Microbiology, 2000, 141-149.
[15] Dierick K, Van Coillie E, Swiecicka I, et al.Fatal family outbreak of Bacillus cereus-associated food poisoning[J].Journal of Clinical Microbiology, 2005, 43 (8) :4277-4279.
[16] 王振国, 刘金华, 肖成蕊, 等.利用PCR技术检测致病性蜡样芽孢杆菌的研究[J].生物技术, 2005, 15 (5) :45-47. [17] 周帼萍, 袁志明.蜡状芽孢杆菌 (Bacillus cereus) 污染及其对食品安全的影响[J].食品科学, 2007, 28 (3) :357-360.
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