Please wait a minute...
浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2018, Vol. 44 Issue (6): 659-666    DOI: 10.3785/j.issn.1008-9209.2017.09.041
生物科学与技术     
单核细胞增生李斯特菌和致病性弧菌的双重肽核酸原位荧光杂交检测
吴姗，李可，方云，楼成杰，虞惠贞，张明哲，帅江冰，张晓峰*
（浙江省检验检疫科学技术研究院，杭州310016）
Simultaneous detection of Listeria monocytogenes and pathogenic Vibrio by dual peptide nucleic acid fluorescence in situ hybridization
WU Shan, LI Ke, FANG Yun, LOU Chengjie, YU Huizhen, ZHANG Mingzhe, SHUAI Jiangbing, ZHANG Xiaofeng*
(Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310016, China)
 全文: PDF(4255 KB)   HTML ( ) 
摘要： 为了同步快速检测单核细胞增生李斯特菌（简称单增李斯特菌）和致病性弧菌，建立了它们的双重肽核酸（peptide nucleic acid, PNA）原位荧光杂交（fluorescence in situ hybridization, FISH）检测方法。在用单重PNA探针分别检测单增李斯特菌和致病性弧菌的基础上，建立了可同步检测它们的双重PNA荧光检测方法，优化了适合这2种菌同步双重杂交的固相荧光原位杂交检测参数。在标记的荧光为FAM和Cy3 的情况下，使用Leica DM 6000B荧光显微镜对4 种荧光滤光模块I3、L5、N21 和Y3 的检测效果进行比较，结果表明：在进行双重荧光检测时，建议选择L5 和Y3 的组合分别观察绿色荧光和红色荧光。比较2 种菌液体积比例对检测结果的影响表明：当比例从1∶1调至1∶9 时，2 种菌可同时在2 个检测通道中被检测到；当比例调至1∶19 时，量少的菌则很难被观察到。综上表明：2 种PNA探针分别被标记上FAM和Cy3 荧光后，可使用Leica DM 6000B荧光显微镜在L5 和Y3 滤镜模块下分别进行绿光和红光的观察。说明若选择合适的荧光及荧光滤光模块，可使用普通荧光显微镜完成单增李斯特菌和致病性弧菌的PNA-FISH同步检测。
关键词： 双重肽核酸探针;  荧光原位杂交;  单核细胞增生李斯特菌;  弧菌    
Abstract: Listeria monocytogenes and pathogenic Vibrio are common and harmful foodborne pathogens in daily life. Besides the culture-based, biochemical, immunological and molecular methods for applications in clinical diagnostics and food microbiology, fluorescence in situ hybridization (FISH) using labeled peptide nucleic acid (PNA) probes as an alternative methodology for detection of L. monocytogenes and Vibrio were developed. To improve the efficiency, multi-probe PNA-FISH assay was established for simultaneous detection of multiple microorganisms. Based on the singlet PNA probe for detection of L. monocytogenes and Vibrio, respectively, by labeling different fluorescent dyes to the specific PNA probes, the PNA-FISH procedure for simultaneous detection of L. monocytogenes and Vibrio was optimized. By the usage of a Leica DM 6000B fluorescence microscope, four kinds of fluorescence filter cubes I3, L5, N21 and Y3 were compared after labeled with FAM and Cy3 fluorescences. As a result, for dual fluorescence detection, the combination of L5 and Y3 was suggested to observe the green fluorescence and red fluorescence, respectively. Comparing the effect of the volume ratio of the two strain solution on the test results, when the ratio decreased from 1∶1 to 1∶9, both strains could be detected; when the ratio decreased to 1∶19, the less was difficult to observe. It is indicated that, after labeled with FAM and Cy3 fluorescences, green and red fluorescences released by PNA probes can be observed through filter cubes L5 and Y3, respectively, within the usage of Leica DM 6000B fluorescence microscope, suggesting that the PNA-FISH synchronously detecting dual-species bacteria can be achieved by ordinary fluorescence microscope, if choosing suitable fluorescent dyes and fluorescence filter cubes.
Key words: dual peptide nucleic acid probes    fluorescence in situ hybridization    Listeria monocytogenes    Vibrio
出版日期: 2018-11-25
CLC:  Q 93-33  
基金资助: 国家重点研发计划（2017YFF0210203）；国家质量监督检验检疫总局科研项目（2013IK189，2016IK284，2017IK206）；浙江省
公益技术应用研究计划（2015C33042）。
通讯作者: 张晓峰（https://orcid.org/0000-0001-6711-9434）     E-mail: breezeyh@hotmail.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
吴姗
李可
方云
楼成杰
虞惠贞
张明哲
帅江冰
张晓峰

引用本文:

吴姗, 李可, 方云, 楼成杰, 虞惠贞, 张明哲, 帅江冰, 张晓峰. 单核细胞增生李斯特菌和致病性弧菌的双重肽核酸原位荧光杂交检测[J]. 浙江大学学报（农业与生命科学版）, 2018, 44(6): 659-666.

WU Shan, LI Ke, FANG Yun, LOU Chengjie, YU Huizhen, ZHANG Mingzhe, SHUAI Jiangbing, ZHANG Xiaofeng. Simultaneous detection of Listeria monocytogenes and pathogenic Vibrio by dual peptide nucleic acid fluorescence in situ hybridization. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(6): 659-666.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2017.09.041    或    http://www.zjujournals.com/agr/CN/Y2018/V44/I6/659

[1] 史亚， 施晓峰， 张永静， 柳景青， 程东庆. 饮用水管网生物膜中弧菌数量影响因素研究[J]. 浙江大学学报(农业与生命科学版), 2016, 42(6): 665-670.
[2] 陈健舜*, 朱凝瑜, 丁雪燕, 姚高华, 陈晓明, 孔蕾, 郑天伦, 何中央. 浙江省主要养殖区凡纳滨对虾（Litopenaeus vannamei）红体病病原研究[J]. 浙江大学学报(农业与生命科学版), 2014, 40(6): 688-696.
[3] 齐学明1， 孙萍2， 吴涛1， 郑晓冬1*, 余挺1*. 浙江省宁波市售血蚶中副溶血性弧菌污染分析及膳食风险评价[J]. 浙江大学学报(农业与生命科学版), 2013, 39(6): 673-678.
[4] 沈锦玉,李新华,潘晓艺,尹文林,郝贵杰. 哈维氏弧菌的主要致病因子及其特性分析[J]. 浙江大学学报(农业与生命科学版), 2011, 37(2): 142-148.