Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (8): 1682-1686.doi: 10.3864/j.issn.0578-1752.2013.08.018

• STORAGE·FRESH-KEEPING·PROCESSING • Previous Articles     Next Articles

Simultaneous Detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae Using Multiplex PCR

 WEI  Shuang, XIAN  Yu-Yin, ZHAO  Hui, WU  Xi-Yang   

  1. 1.Department of Food Science and Engineering, Ji’nan University, Guangzhou 510632
    2.Hubei Entry-Exit Inspection and Quarantine Bureau Inspection and Quarantine Technology Center, Wuhan 430022
    3.Food Safety and Health Emergency Technical Research Center of Guangdong Province, Guangzhou 510632
  • Received:2012-10-26 Online:2013-04-15 Published:2013-01-16

PDF

1322

Cited

9

Abstract: 【Objective】The objective of this study is to develop a multiplex PCR assay that can simultaneously detect Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae in the presence of an internal amplification control (IAC).【Method】Species-specific PCR primers were designed based on gyrB gene for Vibrio alginolyticus, collagenase gene for Vibrio parahaemolyticus, vvhA gene for Vibrio vulnificus and ompW gene for Vibrio cholerae, 16S rRNA gene of bacteria as IAC primers was used to indicate false-negative results. Multiple PCR method was developed after optimization reaction condition. 【Result】The multiple PCR method was proved to be rapid, high-throughput, sensitive and specific and the existence of IAC could successfully eliminate false-negative results. The multiplex PCR was validated with 69 suspicious Vibrio strains and the results were consistent with the physiological and biochemical experiments.【Conclusion】The multiple PCR method is specific, stable and reliable for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae.

Key words: Vibrio , detection , multiple PCR

[1]Toranzo A E, Magarinos B, Romalde J L. A review of the main bacterial fish diseases in mariculture systems. Aquaculture, 2005, 246(1-4): 37-61.

[2]Baffone W, Citterio B, Vittoria E, Casaroli A, Pianetti A, Campana R, Bruscolini F. Determination of several potential virulence factors in Vibrio spp. Isolated from sea water. Food Microbiology, 2001, 18(5): 479-488.

[3]Zhang X H, Austin B. Haemolysins in Vibrio species. Journal of Applied Microbiology, 2005, 98(5): 1011-1019.

[4]Su Y C, Liu C C. Vibrio parahaemolyticus: A concern of seafood safety. Food Microbiology, 2007, 24(6): 549-558.

[5]Schmidt U, Chmel H, Cobbs C. Vibrio alginolyticus infections in humans. Journal of Clinical Microbiology, 1979, 10(5): 666-668.

[6]Vantarakis A, Komninou G, Venieri D, Papapetropoulou. Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels. Letters in Applied Microbiology, 2000, 31(2): 105-109.

[7]Vannuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauters G, Gala J L. Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. Journal of Clinical Microbiology, 1995, 33(11): 2864-2867.

[8]Kong R Y C, Lee S K Y, Law W F, Law S H W, Wu R S S. Rapid detection of six types of bacterial pathogens in mariner waters by multiplex PCR. Water Research, 2002, 36(11): 2802-2812.

[9]Fratamico P M, Sackitey S K, Wiedmann M, Deng M Y. Detection of Escherichia coli O157:H7 by multiplex PCR. Journal of Clinical Microbiology, 1995, 33(8): 2188-2191.

[10]Neogi S B, Chowdhury N, Asakura M, Hinenoya A, Haldar S, Saidi S M, Kogure K, Lara R J, Yamasaki S. A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Letters in Applied Microbiology, 2010, 51(3): 293-300.

[11]Haldar S, Neogi S B, Kogure K, Chatterjee S, Chowdhury N, Hinenoya A, Asakura M, Yamasaki S. Development of a haemolysin gene-based multiplex PCR for simultaneous detection of Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus. Letters in Applied Microbiology, 2010, 50(2): 146-152.

[12]Abualsoud W, Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells. Journal of Clinical Microbiology, 2001, 39(2): 485-493.

[13]Hoorfar J, Malomy B, Abdulmawjood A, Cook N, Wagner M, Fach P. Practical considerations in design of internal amplification controls for diagnostic PCR assays. Journal of Clinical Microbiology, 2004, 42(5): 1863-1868.

[14]Nordstrom J L, Vickery M C L, Blackstone G M, Murray S L, Depaola A. Development of a multoplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Applied and Environmental Micobiology, 2007, 73(18): 5840-5847.

[15]Nandi B, Nandy R K, Mukhopadhyay S, Nair G B, Shimada T, Ghose A C. Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein ompW. Journal of Clinical Microbiology, 2000, 38(11): 4145-4151.

[16]Zhou S, Hou Z, Li N, Qin Q. Development of a SYBR GreenⅠreal-time PCR for quantitative of Vibrio alginolyticus in seawater and seafood. Journal of Applied Microbiology, 2007, 103(5): 1897-1906.

[17]Pinto A D, Ciccarese G, Tantillo G, Catalano D, Forte V T. A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus. Journal of Food Protection, 2005, 68(1): 150-153.

[18]Panicker G, Myers M L, Bej A K. Raped detection of Vibrio vulnificus in shellfish and gulf of mexico water by real-time PCR. Applied and Environmental Microbiology, 2004, 70(1): 498-507.

[19]Izumiya H, Matsumoto K, Yahiro S, Lee J, Morita M, Yamamoto S, Arakawa E, Ohnishi M. Multiplex PCR assay for identification of three major pathogenic Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. Molecular and Cellular Probes, 2011, 25(4): 174-176.

[20]Kim Y B, Okuda J, Matsumoto C, Takahashi N, Hashimoto S, Nishibuchi M. Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene. Journal of Clinical Microbiology, 1999, 37(4): 1173-1177.

[21]Venkatesmwaran K, Dohmoto N, Harayama S. Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp. Applied and Environmental Microbiology, 1998, 64(2): 681-687.

[22]Lee C Y, Chen C H. Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR. Applied and Environmental Microbiology, 1995, 61(4): 1311-1317.

[23]Blackstone G M, Nordstrom J L, Vickery M C L, Bowen M D, Meyer R F, Depala A. Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR. Journal of Microbiological Methods, 2003, 53(2): 149-155.

[24]Pascual J, Macian M C, Arahal D R, Garay E, Pujalte M J. Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes. International Journal of Systematic and Evolutionary Microbiology, 2010, 60(1): 154-165.

[25]吕淑霞, 祝儒刚, 刘月萍, 张喆, 胡英. 扩增内标在副溶血弧菌多重PCR检测方法中的应用. 沈阳农业大学学报, 2010, 41(6): 701-705.

Lü S X, Zhu R G, Liu Y P, Zhang Z, Hu Y. Multiplex PCR assay for the detection of pathogenic Vibrio parahaemolyticus in seafood in the presence of internal amplification control. Journal of Shenyang Agricultural University, 2010, 41(6): 701-705.(in Chinese)
[1] JIAO WenJuan, HE WanLong, GENG HongWei, BAI Bin, LI JianFeng, CHENG YuKun. Stripe Rust Resistance Evaluation and Molecular Characterization of Yr Genes for 155 Spring Wheat Varieties (Lines) [J]. Scientia Agricultura Sinica, 2026, 59(5): 937-950.
[2] GAO YunJie, GUO RuoNan, CHEN ChunWen, DUAN YiFan, ZHANG ZhenJun, NIE ChanChan, LI MengYu, WANG Hui, FENG TingTing, CUI YingYing, DANG GuangHui, LIU SiGuo. Establishment and Preliminary Application of Indirect ELISA Antibody Detection Method for Mycobacterium avium subsp. paratuberculosis in Sheep [J]. Scientia Agricultura Sinica, 2026, 59(3): 655-667.
[3] FU LiJin, CHEN GuanWei, XIAO Gong, WANG XiaoFu, PENG Cheng, CHEN XiaoYun, XU JunFeng, CHEN ZiYan, YANG Lei. A Rapid Detection Method for Genetically Modified Soybean Dbn9004 Based on Dnazyme Signal Amplification [J]. Scientia Agricultura Sinica, 2026, 59(2): 239-249.
[4] LUO Gang, CHENG YiYi, YANG Wen, XIAO YiMeng, YANG ChengXi. CRISPR-Cas12a Gene Editing Technology and Its Application in Agricultural Production [J]. Scientia Agricultura Sinica, 2025, 58(7): 1434-1450.
[5] YAN ChuanBo, GONG Ping, ZHENG WenXin, CHEN XinWen, GUO LeiFeng. Research on Sheep Mounting Behavior Recognition in Complex Scenes Based on an Improved YOLOv11 [J]. Scientia Agricultura Sinica, 2025, 58(18): 3783-3798.
[6] ZHANG XiaoQi, SHEN JianGuo, LIAO FuRong, LI WeiMin, JIN YuJie, WUFUER Shayidan, ZHENG LuPing. Establishment and Application of RT-RAA-CRISPR/Cas12a-Based Visual Detection of Prunus Necrotic Ringspot Virus [J]. Scientia Agricultura Sinica, 2025, 58(12): 2371-2381.
[7] HUANG LiQiang, JIANG Ru, ZHU BoZhi, PENG Huan, XU Chong, SONG JiaXiong, CHEN Min, LI YongQing, HUANG WenKun, PENG DeLiang. Identification and Evaluation of Major Potato Cultivars Resistance to Globodera rostochiensis and Detection of Their H1 Resistance Gene Marker [J]. Scientia Agricultura Sinica, 2024, 57(8): 1506-1516.
[8] CHEN XiHong, CAI Wei, YU Yun, LI Min, WANG NianWu, DU ZhenGuo, SHEN JianGuo, GAO FangLuan. Identification of Tea Plant Viruses in Fujian Province and Establishment of Multiplex PCR Detection Assay [J]. Scientia Agricultura Sinica, 2024, 57(4): 698-710.
[9] ZHAO YiRan, SHAN YanKe, LI JiaHao, HE ZhaoQun, WANG XinYi, WEN Dun, WANG MiLa, CHU Rui, ZHAO DongMing, LIU Fei. Establishment of Rapid Field Co-Detection Method of ASFV Antibody and Nucleic Acid Based on Quantum Dot Microspheres and RPA Technology [J]. Scientia Agricultura Sinica, 2024, 57(24): 4990-5002.
[10] WANG YongJiang, QIAO Qi, WANG Shuang, ZHAO FuMei, TIAN YuTing, ZHANG DeSheng, ZHANG ZhenChen. Establishment and Application of RT-RPA-LFD Detection Method for Sweet Potato Chlorotic Stunt Virus WA Strain [J]. Scientia Agricultura Sinica, 2024, 57(14): 2781-2790.
[11] SU Qi, TANG YaFei, SHE XiaoMan, LAN GuoBing, YU Lin, WU ZhengWei, LI ZhengGang, HE ZiFu. Identification of Viruses Infecting Bottle Gourd in Guangdong Province and Establishment of Multiplex RT-PCR Detection Method [J]. Scientia Agricultura Sinica, 2023, 56(23): 4684-4695.
[12] LI MeiXuan, ZHANG XiangKun, WANG Li, QIAO YueLian, SHI XiaoXin, DU GuoQiang. The Variation of GRSPaV in Different Parts of Shine Muscat Grapevines During Their Phenological Periods [J]. Scientia Agricultura Sinica, 2023, 56(21): 4234-4244.
[13] TANG Wei, ZHANG ChengLing, YANG DongJing, MA JuKui, CHEN JingWei, GAO FangYuan, XIE YiPing, SUN HouJun. Complete Genomic Sequence Characteristics and Establishment of qPCR Detection Technique of Sweet Potato Virus E in China [J]. Scientia Agricultura Sinica, 2023, 56(20): 4010-4020.
[14] SU Jia, ZHAO Wei, LIU Dan, WANG Jia, BAI HongXu, WU HuaWei, XUE QingHong, CHEN XiaoChun. Establishment of Real-Time PCR Method for Detection of Extraneous Marek’s Disease Virus [J]. Scientia Agricultura Sinica, 2023, 56(20): 4125-4136.
[15] LI ZhiLing,LI XiangJu,CUI HaiLan,YU HaiYan,CHEN JingChao. Development and Application of ELISA Kit for Detection of EPSPS in Eleusine indica [J]. Scientia Agricultura Sinica, 2022, 55(24): 4851-4862.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd