食品与发酵工业 >

2020 , Vol. 46 >Issue 10: 271 - 277

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.023146

分析与监测

蜡样芽孢杆菌特异性基因筛选及聚合酶链式反应检测方法的建立

  • 赵岩岩 ,
  • 王书彦 ,
  • 赵琳琳 ,
  • 赵璐 ,
  • 周威 ,
  • 崔震昆 ,
  • 张浩 ,
  • 王淼焱 ,
  • 王书贤 ,
  • 赵圣明
展开
  • 1(河南科技学院 食品学院,河南 新乡,453003)
    2(新乡职业技术学院 旅游管理系, 河南 新乡,453006)
博士,讲师(本文通讯作者,E-mail:zhaoyanyan@hist.edu.cn)

收稿日期: 2019-12-18

  网络出版日期: 2020-06-17

基金资助

河南省高等学校重点科研项目(18B550003);河南省科技发展计划(202102110060;202102110061)

收起

Screening of the specific genes and development of a PCR assay for the detection of Bacillus cereus

  • ZHAO Yanyan ,
  • WANG Shuyan ,
  • ZHAO Linlin ,
  • ZHAO Lu ,
  • ZHOU Wei ,
  • CUI Zhenkun ,
  • ZHANG Hao ,
  • WANG Miaoyan ,
  • WANG Shuxian ,
  • ZHAO Shengming
Expand
  • 1(School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China)
    2(Department of Tourist Management, Xinxiang Vocational and Technology College, Xinxiang 453006, China)

Received date: 2019-12-18

  Online published: 2020-06-17

Fold

摘要

该研究以蜡样芽孢杆菌ATCC 14579的基因组为模板,利用比较基因组学方法筛选并通过聚合酶链式反应(polymerase chain reaction,PCR)验证获得3个蜡样芽孢杆菌的特异性基因,用于食品中蜡样芽孢杆菌的快速检测,并对引物的特异性、灵敏度、抗干扰能力和人工污染检测限等进行评价。结果表明,筛选获得的3个基因片段特异性较好。其中以gene_2626设计的引物gFA2特异性最好,检测灵敏度可达359.5 fg/μL。抗干扰评价结果表明在牛肉背景菌群浓度5.28×107CFU/mL和猪肉背景菌群浓度7.75×106CFU/mL的干扰下,蜡样芽孢杆菌的最低检出限为3.74×103CFU/mL。人工污染试验结果表明,蜡样芽孢杆菌经增菌培养10 h后,人工污染的牛肉和猪肉样品中蜡样芽孢杆菌的检出限分别为4.62和8.38 CFU/g。因此该研究筛选的特异性基因及建立的PCR检测方法在食品中蜡样芽孢杆菌的快速检测方面具有一定的应用潜力。

关键词： 蜡样芽孢杆菌; 特异性基因; 筛选; 聚合酶链式反应

本文引用格式

赵岩岩 , 王书彦 , 赵琳琳 , 赵璐 , 周威 , 崔震昆 , 张浩 , 王淼焱 , 王书贤 , 赵圣明 . 蜡样芽孢杆菌特异性基因筛选及聚合酶链式反应检测方法的建立[J]. 食品与发酵工业, 2020 , 46(10) : 271 -277 . DOI: 10.13995/j.cnki.11-1802/ts.023146

Abstract

Using the genome of Bacillus cereus ATCC 14579 as a template, the specific target of Bacillus cereus was screened by bioinformatics and comparative genomics analysis and three specific genes of Bacillus cereus were identified by PCR protocol. Then, these three specific genes were used for molecular biological detection of Bacillus cereus in food. The primers were evaluated by its specificity, sensitivity, anti-interference capability and artificial contamination. The results showed that three screened primers had better specificity and there was no specific band in non-Bacillus cereus. The primer gFA2 based on 公式had the best sensitivity which reached 359.5 fg/μL. The anti-interference results showed that in the presence of natural background flora enriched from beef (5.28×107 CFU/mL) and pork(7.75×106 CFU/mL)samples, the minimum detection limit of Bacillus cereus was 3.74 × 103 CFU/mL. After 10 h enrichment cultivation of Bacillus cereus, the detection limits of three primers to artificially contaminated beef and pork samples were 4.62 CFU/g and 8.38 CFU/g, respectively. In conclusion, the specific gene screened in this study and the PCR assay could be widely used for rapidly detection of Bacillus cereus in food safety validation.

Key words： Bacillus cereus; specific genes; screening; polymerase chain reaction

参考文献

[1] PIRHONEN T I, ANDERSSON M A, JAASKELAINE E L, et al. Biochemical and toxic diversity of Bacillus cereus in a pasta and meat dish associated with a food-poisoning case[J]. Food Microbiology, 2005, 22(1): 87-91.
[2] VALERO M, HEMANDEZ-HERRERO L A, FEMANDEZ P S, et al. Characterization of Bacillus cereus isolates from fresh vegetables and refrigerated minimally processed foods by biochemical and physiological tests[J]. Food Microbiology, 2002, 19(5): 491-499.
[3] HAN J Z, ZHAO S M, MA Z, et al. The antibacterial activity and modes of LI-F type antimicrobial peptides against Bacillus cereus in vitro[J]. Journal of Applied Microbiology, 2017, 123(3): 602-614.
[4] MOLS M, KRANENBURG R V, PELAARS M H, et al. Comparative analysis of transcriptional and physiological responses of Bacillus cereus to organic and inorganic acid shocks[J]. International Journal of Food Microbiology, 2010, 137(1): 13-21.
[5] TEWARI A, ABDULLAH S. Bacillus cereus food poisoning: International and Indian perspective[J]. Journal of Food Science and Technology, 2014, 52(5): 2 500-2 511.
[6] CETIN K H, NEWMAN M C. Antimicrobial efficacy of phytochemicals against Bacillus cereus in reconstituted infant rice cereal[J]. Food Microbiology, 2018, 2(69):189-195.
[7] BROUSSOLLE V, PANDIANI F, HADDAD N, et al. Insertional mutagenesis reveals genes involved in Bacillus cereus ATCC 14579 growth at low temperature[J]. FEMS Microbiology Letters, 2010, 306(2): 177-183.
[8] WALKLINGRIBEIRO M, RODRÍGUZGONZÁLEZ O, JAYARAM S, et al. Microbial inactivation and shelf life comparison of 'cold' hurdle processing with pulsed electric fields and microfiltration, and conventional thermal pasteurisation in skim milk[J]. International Journal of Food Microbiology, 2011, 144(3): 379-386.
[9] 曹飞扬, 王娉, 江连洲, 等. 蜡样芽孢杆菌分型方法研究进展[J]. 食品科学, 2017, 38(17): 286-290.
[10] 潘娜, 李薇薇, 杨淑香, 等. 中国2002-2015年学校食源性疾病暴发事件归因分析[J]. 中国学校卫生, 2018, 39(4): 570-572.
[11] 黄晶菁, 罗静, 何宏轩. 蜡样芽孢杆菌检测方法研究进展[J]. 中国畜牧兽医, 2018, 45(3): 635-642.
[12] 张志鸿, 许恒毅, 魏华. 基于PCR方法检测蜡样芽孢杆菌的研究进展[J]. 食品工业科技, 2013, 34(22): 335-338.
[13] LI Y, CHEN Q, JIANG H, et al. Novel development of a qPCR assay based on the rpoB gene for rapid detection of Cronobacter spp[J]. Current Microbiology, 2016, 72(4): 436-443.
[14] NAIR M, VENKITANARAYANAN K. Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula[J]. Applied and Environmental Microbiology, 2006, 72(4): 2 539-2 546.
[15] 翟立公, 王俊颖, 张小雨, 等. 甲型副伤寒沙门菌特异性基因筛选及PCR检测体系建立[J]. 食品科学, 2018, 39(2): 151-157.
[16] TAO T T, CHEN Q M, BIE X M, et al. Investigation on prevalence of Listeria spp. and Listeria monocytogenes in animal-derived foods by multiplex PCR assay targeting novel genes[J]. Food Control, 2017,73(PartB):704-711.
[17] 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, 2013, 32(1): 176-185.
[18] MARTÍNE-BLANCH J F, SÁNCHEZ G, GARAY E, et al. Detection and quantification of viable Bacillus cereus in food by RT–qPCR[J]. European Food Research and Technology, 2011, 232: 951-955.
[19] 贾雅菁, 付博宇, 王羽, 等. 实时荧光环介导等温扩增技术检测牛乳中的蜡样芽孢杆菌[J]. 食品科学, 2016, 37(6): 184-189.
[20] CAWTHOM D M, BOTHA S, WITTHUHN R C. Evaluation of different methods for the detection and identification of Enterobacter sakazakii isolated from South African infant formula milks and the processing environment[J]. International Journal of Food Microbiology, 2008, 127(1-2): 129-138.
[21] TAO T T, CHEN Q M, BIE X M, et al. Mining of novel species-specific primers for PCR detection of Listeria monocytogenes based on genomic approach[J]. World Journal of Microbiology and Biotechnology, 2015, 31(12): 1 955-1 966.
[22] 陈启明. 克罗诺杆菌免疫学和分子生物学检测方法研究[D].南京:南京农业大学, 2016:102-103.
[23] PEI X Y, YANG S R, ZhAN L, et al. Prevalence of Bacillus cereus in powdered infant and powdered follow-up formula in China[J]. Food Control, 2018, 93: 101-105.
[24] RENTZEL H, THANH M D, KRAUSE G, et al. Quantification and differentiation of Bacillus cereus group species in spices and herbs by real-time PCR[J]. Food control, 2018, 83: 99-108.
[25] LIU B, ZHOU X J, ZHANG L D, et al. Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis[J]. Food Control, 2012, 27(1): 87-93.
[26] ARUNRUT N, KIATPATHOMCHAI W, ANANCHAIPATTANA C. Multiplex PCR assay and lyophilization for detection of Salmonella spp., Staphylococcus aureus and Bacillus cereus in pork products[J]. Food Science and Biotechnology, 2018, 27: 867-875.
[27] OLIWA-STASIAK K, KOLAJ-ROBIN O, AADLEY C C. Development of real-time PCR assays for detection and quantification of Bacillus cereus group species: Differentiation of B. weihenstephanensis and rhizoid B. pseudomycoides isolates from milk[J]. Applied and Environmental Microbiology, 2011, 77(1): 80-88.
[28] CAAMAÑO-ANTELO S, FERNÁNDEZ NOIE, BÖHME K, et al. Genetic discrimination of foodborne pathogenic and spoilage Bacillus spp. based on three housekeeping genes[J]. Food Microbiology, 2015, 46: 288-298.
[29] WALKERYORKMOORE L, MOORE S C, FOX E M. Characterization of enterotoxigenic Bacillus cereus sensu lato and Staphylococcus aureus isolates and associated enterotoxin production dynamics in milk or meat-based broth[J]. Toxins, 2017, 9(7): 225.
[30] KIM M J, HAN J K, PARK J S, et al. Various enterotoxin and other virulence factor genes widespread among Bacillus cereus and Bacillus thuringiensis strains[J]. Journal of Microbiology and Biotechnology, 2015, 25(6): 872-879.
[31] KAMINSKA P S, YERNAZAROVA A, MURAWSKS E, et al. Comparative analysis of quantitative reverse transcription real-time PCR and commercial enzyme imunoassays for detection of enterotoxigenic Bacillus thuringiensis isolates[J]. FEMS Microbiology Letters, 2014, 357(1): 34-39.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司