环介导等温扩增技术快速检测椰毒假单胞菌的研究
详细信息Rapid detection of Pseudomonas cocovenenans by loop-mediated isothermal amplification
-
摘要: 建立环介导等温扩增技术(LAMP)快速检测椰毒假单胞菌的方法。根据公布的椰毒假单胞菌16S～23SrRNA基因序列设计引物,建立了LAMP反应体系,在此基础上检测了蜡样芽孢杆菌菌液和人工污染蜡样芽孢杆菌的银耳样品,并将LAMP法与PCR法进行比较。结果表明,LAMP检测方法具有较高的特异性和敏感性,椰毒假单胞菌的检出限为5.4CFU/mL,是PCR方法检测灵敏度的1000倍,人工污染银耳样品中的椰毒假单胞菌的检出限为76CFU/g,样品中椰毒假单胞菌的检测过程(包括DNA提取、LAMP和电泳)可在2h内完成,因此LAMP可以简便快速有效地检测食品中的椰毒假单胞菌。Abstract: An assay using loop-mediated isothermal amplification ( LAMP) technology was developed for rapid detection of Pseudomonas cocovenenans. The 16S ～ 23S rRNA internal transcribed spacer ( ITS) sequence of Pseudomonas cocovenenans as target sequences, used to design LAMP primers.The LAMP mixture was made in 25μL of reaction mixture, also LAMP was used both in Pseudomonas cocovenenans and in tremella of Pseudomonas cocovenenans and we compaired the method with PCR.The result showed that a rapid technique for detection of Pseudomonas cocovenenans initially established by LAMP. The sensitivity of LAMP assay was 1000 times as high as PCR, the sensitivity of the LAMP was 5.4CFU /mL, and the detection limit of artificially contaminated tremella was 76CFU /g. The whole experimental procedure ( contain DNA purified, LAMP and UV transilluminator) can be completed in 2h. LAMP technology provides a simple, rapid and sensitive method for detection Pseudomonas cocovenenans in food.
-
[1] 王静, 刘秀梅.椰毒假单胞菌酵米面亚种及米酵菌酸的研究进展 (综述) [J].中国食品卫生杂志, 1996, 8 (2) :42-45. [2] 刘秀梅.我国食品微生物微生物学30年来的热点研究及主要进展[J].中国食品卫生杂志, 2009, 21 (4) :289-294. [3] 赵乃昕.酵米面中毒病原菌——椰毒假单胞菌.潍坊医学院学报[J].1990, 1 (2) :78-82. [4] 郭铃.椰毒假单胞菌食物中毒实验室检测.现代预防医学[J].2007, 34 (6) :1145-1147. [5] 林黎明, 赵乃昕.导数光谱谱线组判别分析法鉴别椰毒假单胞菌[J].中国公共卫生, 2002, 18 (7) :860-861. [6] 邱茂峰, 刘秀梅, 杨瑞馥.用rDNA指纹图分析我国部分椰酵假单胞菌的分子流行病学特征[J].卫生研究, 1998, 27 (1) :57-60. [7] JIAO Zhen-quan, CAO Wei, YU Dong-min, et al.Study on Comparison of16S-23S rRNA Gene ISR Sequence of Pseudomonas cocovenenans subsp.farinofermentans Strains and Burkholderia gladioli Strains[J].China Journal of Food Hygiene, 2008, 20 (3) :197-203.
[8] Notomi T, Okayama H, Masubuchi H, et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res, 2000, 28 (12) :E63.
[9] 李玉锋, 李帅, 黄丽娟, 等.LAMP技术在食品致病菌检测中的研究进展.[J].西华大学学报:自然科学版, 2011, 30 (1) :16-18. [10] 张胜凯, 马晓燕, 张先舟, 等.环介导等温扩增技术快速检测食品中变形杆菌的研究[J].食品工业科技, 2011, 32 (6) :384-387. [11] 刘哲, 马晓燕, 张会彦, 等.环介导等温扩增技术快速检测产气荚膜梭菌的研究[J].中国食品学报, 2012, 12 (4) :168-173.
计量
- 文章访问数: 115
- HTML全文浏览量: 8
- PDF下载量: 206
下载:
