Citation: Lin Peng, Lu Jie, Guo Songlin, Feng Jianjun, Wang Yilei, Chen Jinmin. Sensitive Detection of Aeromonas Hydrophila by Time-Resolved Fluorimmunoassay[J]. Acta Chimica Sinica, ;2017, 75(4): 398-402. doi: 10.6023/A16110619 shu

Sensitive Detection of Aeromonas Hydrophila by Time-Resolved Fluorimmunoassay

Fisheries College, Jimei University, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Xiamen 361021

Corresponding author: Lin Peng, linpeng@jmu.edu.cn
Received Date: 21 November 2016

Fund Project: the National Natural Science Foundation of China 31272685

Figures(4)

The aim of this study is to establish a biotin-avidin system and time-resolved fluorimmunoassay (BAS-TRFIA) for quantitative analysis of Aeromonas Hydrophila. First, A. Hydrophila strain B18 isolated from diseased Anguilla rostrata, was used for rabbit anti-serum preparation by injecting the formalin-inactivated bacteria. The rabbit immunoglobulin (IgG) was purified by SPA affinity column. Then, a 96-well microtiter plate precoated with the rabbit IgG was incubated with the strain B18, and biotinylated IgG was pipette to the wells to form a typical double-antibody-sandwich immune complex. Finally, Eu³⁺-labeled streptavidin was added the wells to produce affinity reaction. Time-resolved fluorescence (TRF) of Eu³⁺ was measured in enhancement solution to reflect the quantity of the strain B18. This assay showed a good relationship between TRF and the concentration of the strain B18. The limit of detection (LOD) was 1.0×10² cfu/mL, and the LOD of BAS-TRFIA was improved 400-fold compared to the already reported ELISA. The established sandwich BAS-TRFIA showed a good sixth order polynomial fitting from 1.0×10 to 1.0×10⁶ cfu/mL for the strain B18 with the correlation coefficient 0.9716. The strain B18 was positive in BAS-TRFIA, while 19 other contrast strains of Aeromonas eucrenophila, Aeromonas jandaei, Aeromonas enteropelogenes, Escherichia coli, Aeromonas bestiarum and Aeromonas media etc. were negative in the assay, indicating that the antibody had high specificity. The intra-assay and inter-assay standard deviation were less than 5.00% and 9.00%, respectively. The TRF didn't change obviously after the related reagents were placed at 37℃ for 6 days. The method was used to detect the tissues including liver, kidney, intestine, gill and muscle from A. rostrata infected artificial with the strain B18. The result showed 98.33% of 60 samples were positive. BAS-TRFIA for A. hydrophila strain B18 was sensitive, specific and simple. The results indicate that the established BAS-TRFIA has potential application for screening A. hydrophila in aquaculture.
- time-resolved fluorimmunoassay,
- Aeromonas hydrophila,
- biotin-avidin system,
- Anguilla rostrata,
- aquaculture
1. [1]
  Meng, S.; Wang, Y.; Wang, Y.; Liu, D.; Ye, C. Lett. Appl. Microbiol. 2015, 61(2), 171.
2. [2]
  Chalifoux, L. V.; Hajema, E. M.; Leeparritz, D. Lab. Anim. Sci. 1993, 43(4), 355.
3. [3]
  Fu, X. Z.; Lin, Q.; Liu, L. H.; Liang, H. R.; Huang, Z. B.; Li, N. Q. Fish Shellfish Immun. 2016, 56, 286. doi: 10.1016/j.fsi.2016.07.023
4. [4]
  Griffin, M. J.; Goodwin, A. E.; Merry, G. E.; Liles, M. R.; Wil-liams, M. A.; Ware, C.; Waldbieser, G. C. J. Vet. Diagn. Invest. 2013, 25(4), 473. doi: 10.1177/1040638713494210
5. [5]
  Guo, S.-L.; Guan, R.-Z. Journal of Huazhong Agriculture University 2009, 28(4), 463(in Chinese).
6. [6]
  Chauret, C.; Volk, C.; Creason, R.; Jarosh, J.; Robinson, J.; Warnes, C. Can. J. Microbiol. 2001, 47(8), 782.
7. [7]
  Hussain, I. A.; Jeyasekaran, G.; Shakila, R. J.; Raj, K. T.; Jeevithan, E. J. Food Sci. Tech. Mys. 2014, 51(2), 401. doi: 10.1007/s13197-013-1190-9
8. [8]
  Chen, C.-F. J. Huazhong Agriculture University 1998, 17(3), 264(in Chinese).
9. [9]
  Hu, D.-Y.; Qian, D.; Liu, W.; Zhang, Y.-P.; Pan, Q.-Q.; Chen, C.-F. Journal of Huazhong Agriculture University 2008, 27(1), 80(in Chinese).
10. [10]
  Jiang, H. F.; Wang, G. L.; Zhang, W. Z.; Liu, X. Y.; Ye, Z. Q.; Jin, D. Y.; Yuan, J. L.; Liu, Z. G. J. Fluoresc. 2010, 20(1), 321. doi: 10.1007/s10895-009-0559-7
11. [11]
  Kupstat, A.; Kumke, M. U.; Hildebrandt, N. Analyst 2011, 136(5), 1029. doi: 10.1039/C0AN00684J
12. [12]
  Lu, J.; Guo, S.-L.; Feng, J.-J.; Wang, Y.-L.; Zhu, H.-Q.; Lin, P. Chinese J. Anal. Chem. 2012, 40(11), 1709(in Chinese).
13. [13]
  Huang, Y. H.; Xu, Y.; He, Q. H.; Chu, J. S.; Du, B. B.; Liu, J. Food Agr. Immunol. 2014, 25(2), 186. doi: 10.1080/09540105.2012.759540
14. [14]
  Tang, J.; Huang, Y. P.; Liu, H. Q.; Zhang, C. C.; Tang, D. P. Biosens. Bioelectron. 2016, 79, 508. doi: 10.1016/j.bios.2015.12.097
15. [15]
  Zhu, L.-P.; Chen, X.-Q. Experimental Methods Used in Immunology, People's Military Medical Press, Beijing, 2000, pp. 18~22(in Chinese).
16. [16]
  Zheng, W.-F. Medical Immunology, People's Medical Publishing House, Beijing, 1989, p. 38(in Chinese).
17. [17]
  Shen, J.; Lin, D.-Q.; Xu, J. Chinese Bulletin of Life Science 2004, 16(1), 55(in Chinese).
18. [18]
  Li, W.-H.; He, X.-W.; Qi, W.; Chen, F.-Y. Modern Food Science & Technology 2011, 27(12), 1480(in Chinese).
1. [1]
  Xinting XIONG , Zhiqiang XIONG , Panlei XIAO , Xuliang NIE , Xiuying SONG , Xiuguang YI . Synthesis, crystal structures, Hirshfeld surface analysis, and antifungal activity of two complexes Na(Ⅰ)/Cd(Ⅱ) assembled by 5-bromo-2-hydroxybenzoic acid ligands. Chinese Journal of Inorganic Chemistry, 2024, 40(9): 1661-1670. doi: 10.11862/CJIC.20240145
2. [2]
  Xiaoning TANG , Junnan LIU , Xingfu YANG , Jie LEI , Qiuyang LUO , Shu XIA , An XUE . Effect of sodium alginate-sodium carboxymethylcellulose gel layer on the stability of Zn anodes. Chinese Journal of Inorganic Chemistry, 2024, 40(8): 1452-1460. doi: 10.11862/CJIC.20240191
3. [3]
  Xin MA , Ya SUN , Na SUN , Qian KANG , Jiajia ZHANG , Ruitao ZHU , Xiaoli GAO . A Tb₂ complex based on polydentate Schiff base: Crystal structure, fluorescence properties, and biological activity. Chinese Journal of Inorganic Chemistry, 2024, 40(7): 1347-1356. doi: 10.11862/CJIC.20230357
4. [4]
  Jinlong YAN , Weina WU , Yuan WANG . A simple Schiff base probe for the fluorescent turn-on detection of hypochlorite and its biological imaging application. Chinese Journal of Inorganic Chemistry, 2024, 40(9): 1653-1660. doi: 10.11862/CJIC.20240154
5. [5]
  Kexin Dong , Chuqi Shen , Ruyu Yan , Yanping Liu , Chunqiang Zhuang , Shijie Li . Integration of Plasmonic Effect and S-Scheme Heterojunction into Ag/Ag₃PO₄/C₃N₅ Photocatalyst for Boosted Photocatalytic Levofloxacin Degradation. Acta Physico-Chimica Sinica, 2024, 40(10): 2310013-. doi: 10.3866/PKU.WHXB202310013
6. [6]
  Yonghui ZHOU , Rujun HUANG , Dongchao YAO , Aiwei ZHANG , Yuhang SUN , Zhujun CHEN , Baisong ZHU , Youxuan ZHENG . Synthesis and photoelectric properties of fluorescence materials with electron donor-acceptor structures based on quinoxaline and pyridinopyrazine, carbazole, and diphenylamine derivatives. Chinese Journal of Inorganic Chemistry, 2024, 40(4): 701-712. doi: 10.11862/CJIC.20230373
7. [7]
  Zeyu XU , Anlei DANG , Bihua DENG , Xiaoxin ZUO , Yu LU , Ping YANG , Wenzhu YIN . Evaluation of the efficacy of graphene oxide quantum dots as an ovalbumin delivery platform and adjuvant for immune enhancement. Chinese Journal of Inorganic Chemistry, 2024, 40(6): 1065-1078. doi: 10.11862/CJIC.20240099
8. [8]
  Chunmei GUO , Weihan YIN , Jingyi SHI , Jianhang ZHAO , Ying CHEN , Quli FAN . Facile construction and peroxidase-like activity of single-atom platinum nanozyme. Chinese Journal of Inorganic Chemistry, 2024, 40(9): 1633-1639. doi: 10.11862/CJIC.20240162
9. [9]
  Di WU , Ruimeng SHI , Zhaoyang WANG , Yuehua SHI , Fan YANG , Leyong ZENG . Construction of pH/photothermal dual-responsive delivery nanosystem for combination therapy of drug-resistant bladder cancer cell. Chinese Journal of Inorganic Chemistry, 2024, 40(9): 1679-1688. doi: 10.11862/CJIC.20240135
10. [10]
  Siyi ZHONG , Xiaowen LIN , Jiaxin LIU , Ruyi WANG , Tao LIANG , Zhengfeng DENG , Ao ZHONG , Cuiping HAN . Targeting imaging and detection of ovarian cancer cells based on fluorescent magnetic carbon dots. Chinese Journal of Inorganic Chemistry, 2024, 40(8): 1483-1490. doi: 10.11862/CJIC.20240093
11. [11]
  Haitang WANG , Yanni LING , Xiaqing MA , Yuxin CHEN , Rui ZHANG , Keyi WANG , Ying ZHANG , Wenmin WANG . Construction, crystal structures, and biological activities of two Ln^Ⅲ₃ complexes. Chinese Journal of Inorganic Chemistry, 2024, 40(8): 1474-1482. doi: 10.11862/CJIC.20240188
12. [12]
  Peiran ZHAO , Yuqian LIU , Cheng HE , Chunying DUAN . A functionalized Eu³⁺ metal-organic framework for selective fluorescent detection of pyrene. Chinese Journal of Inorganic Chemistry, 2024, 40(4): 713-724. doi: 10.11862/CJIC.20230355
13. [13]
  Jiakun BAI , Ting XU , Lu ZHANG , Jiang PENG , Yuqiang LI , Junhui JIA . A red-emitting fluorescent probe with a large Stokes shift for selective detection of hypochlorous acid. Chinese Journal of Inorganic Chemistry, 2024, 40(6): 1095-1104. doi: 10.11862/CJIC.20240002
14. [14]
  Zhaoyang WANG , Chun YANG , Yaoyao Song , Na HAN , Xiaomeng LIU , Qinglun WANG . Lanthanide(Ⅲ) complexes derived from 4′-(2-pyridyl)-2, 2′∶6′, 2″-terpyridine: Crystal structures, fluorescent and magnetic properties. Chinese Journal of Inorganic Chemistry, 2024, 40(8): 1442-1451. doi: 10.11862/CJIC.20240114

Get Citation

PDF

XML

Metrics

PDF Downloads(8)
Abstract views(1352)
HTML views(76)

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142