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| 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) |
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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.
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Published: 25 November 2018
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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
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
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