食品科学 ›› 2024, Vol. 45 ›› Issue (9): 219-225.doi: 10.7506/spkx1002-6630-20230621-169

• 安全检测 • 上一篇    下一篇

基于体温扩增的唐菖蒲伯克霍尔德氏菌椰毒致病变种3种可视化快速检测方法的建立

林志伟,王帅,蔡杰,聂丹丹,李涛,李红娜,杨艳歌,袁飞   

  1. (1.黑龙江八一农垦大学农学院,黑龙江 大庆 163000;2.中国检验检疫科学研究院,国家市场监管重点实验室(食品质量与安全),北京 100176;3.黑龙江八一农垦大学生命科学技术学院,黑龙江 大庆 163000;4.长春海关技术中心,吉林 长春 130062)
  • 出版日期:2024-05-15 发布日期:2024-05-01
  • 基金资助:
    国家市场监督管理总局技术保障专项(2023YJ17);“十四五”国家重点研发计划重点专项(2022YFF0607900)

Establishment of Three Rapid Visual Detection Methods for Burkholderia gladioli pv. cocovenenans Based on Body Temperature Amplification

LIN Zhiwei, WANG Shuai, CAI Jie, NIE Dandan, LI Tao, LI Hongna, YANG Yange, YUAN Fei   

  1. (1. College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing 163000, China; 2. Key Laboratory of Food Quality and Safety for State Market Regulation, Chinese Academy of Inspection and Quarantine, Beijing 100176, China; 3. College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing 163000, China; 4. Changchun Customs Technical Center, Changchun 130062, China)
  • Online:2024-05-15 Published:2024-05-01

摘要: 为实现食品中唐菖蒲伯克霍尔德氏菌椰毒致病变种(Burkholderia gladioli pv. cocovenenans)的快速检测,基于体温扩增技术——酶促重组等温扩增(enzymatic recombinase amplification,ERA),并根据唐菖蒲伯克霍尔德氏菌椰毒致病变种bonM基因序列设计和筛选了ERA检测引物和探针,分别建立ERA显色法、荧光法、试纸条法3 种可视化快速检测方法,并评估其特异性和灵敏度,以及在市售样品检测适用性和准确性。结果显示,建立的方法对3 株唐菖蒲伯克霍尔德氏菌椰毒致病型菌株均有扩增,其他常见的食源性致病菌以及其他唐菖蒲伯克霍尔德氏菌均无扩增,说明检测方法的特异性良好。3 种方法的检出限均为10-2 ng/μL,灵敏度较好。采用建立的3 种可视化快速检测方法对15 份市售样品进行检测,检出阳性2 份,检出率为13.3%。该结果与国标方法的检测结果一致,说明方法具有较好的适用性和准确性。本研究建立的基于体温扩增技术的显色法、荧光法和试纸条法具有较高的特异性及灵敏度,37 ℃体温扩增15 min左右即可获取检测结果,且裸眼可视,可为食品中唐菖蒲伯克霍尔德氏菌椰毒致病变种的现场可视化快速筛查提供新型简便的策略。

关键词: 唐菖蒲伯克霍尔德氏菌椰毒致病变种;酶促等温扩增技术;可视化;快速检测

Abstract: Three rapid visual methods, namely chromogenic, fluorescence and test strip, for the rapid detection of Burkholderia gladioli pv. cocovenenans in foods were established based on enzymatic recombinase amplification (ERA). Primers and probes were designed and screened based on the bonM gene of B. gladioli pv. cocovenenans. Then the specificity and sensitivity of the three methods were evaluated, as well as their applicability and accuracy in the detection of commercial food samples. The results showed that three strains of B. gladioli pv. cocovenenans, but not other common foodborne pathogens and other B. gladioli strains, were amplified by the three methods, indicating their good specificity. The detection limits of these methods were all 10-2 ng/μL, and their sensitivity was good. Out of 15 commercial samples, two tested positive by each of these methods with a detection rate of 13.3%. This result was consistent with that of the national standard method, indicating that our methods had good applicability and accuracy. All three methods give results that can be observed by the naked eye after amplification at 37 ℃ for 15 min, which provide a new and simple strategy for the rapid, visual and on-site screening of B. gladioli pv. cocovenenans in foods.

Key words: Burkholderia gladioli pv. cocovenenans; enzymatic recombinase amplification; visualization; rapid detection

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