Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology  2010, Vol. 30 Issue (06): 96-102    DOI: Q819
    
Establishment of Rapid Detection Method for Vibrio vulnificus in Aquatic Food by Loop-Mediated Isothermal Amplification
XU Yi-gang1,LI Su-long1,YANG Jun-hong3,LI Dan-dan4,JIANG Yan-chun1,XIE Xiao-feng1,CUI Li-chun2
1.Technical Centre of Heilongjiang Entryexit Inspection and Quarantine Bureau, Harbin 150001,China
2.Northeast Forestry University, Harbin 150040,China
3.Harbin Grain Research Institute, Harbin 150076,China
4.Technical Centre of Hainan Entry-exit Inspection and Quarantine Bureau, Haikou 570125,China
Download: HTML   PDF(811KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

Vibrio vulnificus is an important foodborne pathogen, which mainly exists in estuaries and the marine environment and seriously harms aquaculture development and human health. Therefore, the development of a technique that can identify the pathogen quickly, accurately and protect aquaculture development and enhance the food safety is of great importance. Here a rapid method for detecting Vibrio vulnificus with loop-mediated isothermal amplification (LAMP) using the vvHA gene of Vibrio vulnificus as target gene were established. Forty-six bacterial strains were selected to test specificity of the LAMP method and only Vibrio vulnificus strain was LAMP positive result, and indicating the high specificity of the LAMP method. The LAMP method could be completed in 40~60min and showed a good sensitivity of 15 CFU/ml for cultured Vibrio vulnificus and of 20 CFU/g for its contaminated food. Finally, 25 samples in detected 278 practical samples of shrimps, crabs, oysters and artificial contamination ones were positive results using the LAMP method, which accorded with the detection results according to SN/T 1870-2007. The LAMP assay is a sensitive, rapid simple tool for the detection of Vibrio vulnificus.



Key wordsVibrio vulnificus      Foodborne pathogen      Loop-mediated isothermal amplification (LAMP)      Rapid detection     
Received: 28 January 2010      Published: 12 June 2010
Cite this article:

XU Xi-Gang, LI Su-Long, YANG Jun-Hong, LI Dan-Dan, JIANG Yan-Chun, XIE Xiao-Feng, CUI Li-Chun. Establishment of Rapid Detection Method for Vibrio vulnificus in Aquatic Food by Loop-Mediated Isothermal Amplification. China Biotechnology, 2010, 30(06): 96-102.

URL:

https://manu60.magtech.com.cn/biotech/Q819     OR     https://manu60.magtech.com.cn/biotech/Y2010/V30/I06/96

[1] Bisharat N, Cohen D I, Harding R M, et al. Hybrid Vibrio vulnificus. Emerg Infect Dis, 2005, 11(1): 3035. 
[2] Chiang S R, Chuang Y C. Vibrio vulnificus infection: clinical manifestations, pathogenesis, and antimicrobial therapy. J Microbiol Immunol Infect, 2003, 36(2): 8188. 
[3] Ruppert J, Panzig B, Guertler L, et al. Two cases of severe sepsis due to Vibrio vulnificus wound infection acquired in the Baltic Sea. Eur J Clin Microbiol Infect Dis, 2004, 23: 912915. 
[4] Chung P H, Chuang S K, Tsang Y, et al. Cutaneous injury and Vibrio vulnificus infection. Emerg Infect Dis, 2006, 12 (8): 13021303. 
[5] 卢中秋, 李秉熙, 黄唯佳,等. 创伤弧菌败血症的临床和流行病学特点. 中华预防医学杂志, 2003, 37 (5): 378. Lu Z Q, Li B X, Huang W J, et al.Clin J Prev Med, 2003, 37 (5): 378. 
[6] 周建芳, 施玉铭, 王志刚. 创伤弧菌感染患者的护理. 解放军护理杂志, 2008, 5(6A): 4445. Zhou J F, Shi Y M, Wang Z G.Nurs J Chin PLA, 2008, 5(6A): 4445. 
[7] 吴碎秋,练红,龚裕强,等. 原发性创伤弧菌败血症的护理21例. 中国实用护理杂志, 2005, 21(11A): 1516. Wu S Q, Lian H, Gong Y Q.Chin J Prac Nurs, 2005, 21(11A): 1516. 
[8] Underwood J, Sperling J D, Flomenbaum N E, et al. A fatal case of Vibrio vulnificus septicemia from a nongulf state: A public health alert for patients with chronic liver disease. Am J Emerg Med, 2006, 24 (9): 621624. 
[9] 马骢, 郭建巍, 郝秀红,等.中国海域分离的创伤弧菌的致病性及其对抗生素的敏感性研究. 第四军医大学学报, 2009, 30(6): 493496. Ma C, Guo J W, Hao X H, et al.J Fourth Mil Med Univ, 2009, 30(6): 493496. 
[10] Hsueh P R, Lin C Y, Tang H J, et al. Vibrio vulnificus in Taiwan. Emerg Infect Dis, 2004, 10(8): 13631368. 
[11] Humar H S, Parvathi A, Karunasagar I. et al. A gyrBbased PCR for the detection of Vibrio vulnificus and its application for direct detection of this pathogen in oyster enrichment broths. Int J Food Microbiol, 2006 (3): 216220. 
[12] Bauer A, RΦrvik L M. A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. Lett Appl Microbiol, 2007, 45 (4): 371375. 
[13] Campbell M S, Wright A C. Realtime PCR analysis of Vibrio vulnificus from oysters. Appl Environ Microbiol, 2003, 69(12): 71377144. 
[14] Panicker G, Bej A K. Realtime PCR detection of Vibrio vulnificusin oysters: comparison of oligonucleotide primers and probes targeting vvhA. Appl Environ Microbiol, 2005, 71(10): 57025709. 
[15] Notomi T, Okayama H, Masubuchi H, et al. Loopmediated isothermal amplification of DNA. Nucleic Acids Res, 2000, 28(12):6363. 
[16] Mori Y, Nagamine K, Tmita N, et al. Detection of Loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem and Biophys Res Commun, 2001, 289: 150154. 
[17] Nagamine K, Watanabe K, Ohtsuka K, et al. Loopmediated isothermal amplification reaction using a nondenatured template. Clin Chem, 2001, 47: 17421743. 
[18] Song T, Toma C, Nakasone N, et al. Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loopmediated isothermal amplification method. FEMS Microbiol Lett, 2005, 243: 259263. 
[19] Enomoto Y, Yoshikawa T, Ihira M, et al. Rapid diagnosis of herpes simplex virus infection by a loopmediated isothermal amplification method. J Clin Microbiol, 2005, 43(2): 951955. 
[20] Amazaki W, Taguchi M, Ishibashi M, et al. Development and evaluation of a loopmediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli. J Clin Microbiol, 2008, 57: 444451. 
[21] Osaka K, Komatsuzaki M, Takahashi H, et al. Vibrio vulnificus septicaemia in Japan: an estimated number of infections and physicians’knowledge of the syndrome. Epidemiol Infect, 2004, 132(5): 993996. 
[22] Harwood V J, Gandhi J P, Wright A C. Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources: a review. J Microbiol Methods, 2004, 59(3):301316. 
[23] Cerda C M, Jofre J, Blanch A R. A selective medium and a specific probe for detection of Vibrio vulnificus. Appl Environ Microbiol, 2000, 66 (2): 855859. 
[24] Cerda C M, Permin L, Larsen J L. et al. Comparison of selective media for the detection of Vibrio vulnificus in environmental samples. J Appl Microbiol, 2001, 91(2):322327. 
[25] Parker R W, Lewis D H. Sandwich enzymelinked immunosorbent assay for Vibrio vulnificus hemolysin to detect V. vulnificusin environmental specimens. Appl Environ Microbiol, 1995, 61(2): 476480.

[1] ZHOU Zi-hui,LIU Xiao-xian,HUANG Hao,XIAO Rui,QI Ke-zong,WANG Sheng-qi. Application of Surface-enhanced Raman Scattering Based onNano-signal Tags in Pathogen Detection[J]. China Biotechnology, 2021, 41(2/3): 70-77.
[2] HUANG Hai-long, ZHU Peng, YANG Hao. LAMP-LFD Technology and Its Application in Rapid Detection of Biological[J]. China Biotechnology, 2015, 35(12): 89-95.
[3] FENG Fei, XIE Zhen-wen, ZENG Mu-heng. Studies on Detection of Salmonella typhimurium in Food by Multiplex Polymerase Chain Methods[J]. China Biotechnology, 2011, 31(01): 65-69.
[4] . Development of Loop-Mediated Isothermal Amplification ( LAMP ) Method for Detection of Vibrio parahaemolyticus[J]. China Biotechnology, 2007, 27(12): 66-72.