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Development of a SYBR Green I real-time PCR for detection of Vibrio harveyi based on the vhhP2 gene
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1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences; Key Laboratory of Susainable De-velopment of Marine Fisheries, Ministry of Agriculture, Qingdao 266071, China;2. Shandong Entry-Exit Inspection and Quarantine Bureau of the P

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S942

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    Abstract:

    is an important pathogenic bacterium of most marine aquaculture animals that has caused significant losses within the aquaculture industry. The pathogenicity of is influenced by quorum sensing, meaning that population density plays a role in determining the outcome of an infection. Thus, there is a need to develop a method to detect the density of V. harveyi species-specificP2 gene to establish a SYBR Green I real-time fluorescence quantitative PCR detection method. A 151 bp gene fragment was amplified from the chromosomal DNA of from different sources. The primers did not cross react with nine other bacteria species using conventional PCR, suggesting the primer pair has good intra-species specificity and inter-species commonality. A recombinant plasmid containing the was constructed and used to construct the standard curve. The standard curve for the +37.48. The correlation coefficient was 0.998 and the amplification efficiency was 1.00, indicating that there was a good linear relationship between initial templates and values. The melting curve had only one specific peak at an annealing temperature of 60. The detection limit of the assay was seven copies per reaction, which is 10 000 times more sensitive than that of conventional polymerase chain reaction (PCR). The results of intra- and inter-assay variability tests demonstrated that the method was highly reproducible. Our results suggest that this SYBR Green I real-time PCR assay may be used for the rapid and accurate detection of from infected aquaculture species. This will allow early diagnosis of infection and improve the efficacy of disease prevention and surveillance programs.

    Reference
    [1] Pizzutto M, Hirst R G. Classification of isolates of Vibrio harveyi virulent to Penaeus monodon larvae by protein profile analysis and M 13 DNA fingerprinting[J]. Dis Aquat Organ, 1995, 21(1): 61−68.
    [2] Alvarez J D, Austin B, Alvarez A M, et al. Vibrio harveyi: a pathogen of penaeid shrimps and fish in Venezuela[J]. J Fish Dis, 1998, 21(4): 313−316.
    [3] Wang B K, Yu J H, Li J, et al. Isolation and identification of pathogen ( Vibrio harveyi)from sea perch , Lateolabrax ja-ponicus[J]. Journal of Fishery Sciences of China, 2002, 9(1): 52−55.王保坤, 余俊红, 李筠, 等. 花鲈弧菌病病原菌(哈维氏弧菌)的分离与鉴定[J]. 中国水产科学, 2002, 9(1): 52−55.
    [4] Xu X J, Xu B, Wang J, et al. Studies on blood chemistry indices and histopathology of Pseudosciaena crocea artifi-cially challenged with Vibrio harveyi[J]. Journal of Fisheries of China, 2010, 34(4): 618−625.徐晓津, 徐斌, 王军, 等. 大黄鱼感染哈维氏弧菌后血液生化指标的变化及组织病理学观察[J]. 水产学报, 2010, 34(4): 618−625.
    [5] Li N Q, Bai J J, Wu S Q, et al. Molecular classification of three kinds of pathogenic Vibrios in orange-spotted grouper, Ep-inephelus coioides[J]. Journal of Fisheries of China, 2005, 29(3): 356−361.李宁求, 白俊杰, 吴淑勤, 等. 斜带石斑鱼 3 种致病性弧菌的分子生物学鉴定[J]. 水产学报, 2005, 29(3): 356−361.
    [6] Fan W H, Huang J, Wang X H, et al. Identification and phy-logenetic study of pathogenic bacteria causing ulcer disease of cultured Turbot ( Scophthalmus maximus)[J]. Acta Mi-crobiologica Sinica, 2005, 45(5): 665−670.范文辉, 黄倢, 王秀华, 等. 养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析[J]. 微生物学报, 2005, 45(5): 665−670.
    [7] Song J F, Huang W F, Zhang J Y. Study on the pathogen of hemorrhage disease of Paralichthys olivaceus[J]. Journal of Microbiology, 2004, 24(2): 51−53.宋靖芳, 黄文芳, 张剑英. 牙鲆出血病病原的初步研究[J]. 微生物学杂志, 2004, 24(2): 51−53.
    [8] Oakey H J, Levy N, Bourne D G, et al. The use of PCR to aid in the rapid identification of Vibrio harveyi isolates[J]. J Appl Microbiol, 2003, 95(6): 1293−1303.
    [9] Fukui Y, Sawabe T. Rapid detection of Vibrio harveyi in seawater by real-time PCR[J]. Microbes Environ, 2008, 23(2): 172−176.
    [10] Conejero M J U, Hedreyda C T. Isolation of partial toxR gene of Vibrio harveyi and design of toxR targeted PCR primers for species detection[J]. J Appl Microbiol, 2003, 95(3): 602−611.
    [11] Pang L, Zhang X H, Zhong Y, et al. Identification of Vibrio harveyiusing PCR amplification of the toxR gene[J]. Lett Appl Microbiol, 2006, 43(3): 249−255.
    [12] Conejero M J U, Hedreyda C T. PCR detection of hemolysin (vhh) gene in Vibrio harveyi[J]. J Gen Appl Microbiol, 2004, 50(3): 137−142.
    [13] Robertson P A W, Xu H S, Austin B. An enzyme-linked immunosorbent assay (ELISA) for the detection of Vibrio harveyi in penaeid shrimp and water[J]. JMicrobiol Meth, 1998, 34(1): 31−39.
    [14] Austin B, Austin D A, Farhat Z, et al. Diagnosis and control of bacterial fish pathogens with emphasis on Vibrio Harveyi[J]. Jornal of Ocean University of Qingdao, 1999, 29(3): 489−499.以哈维氏弧菌(Vibrio harveyi)为重点的鱼类细菌病原的诊断与控制(英文) [J]. 青岛海洋大学学报:自然科学版, 1999, 29(3): 489−499.
    [15] Miller M B, Bassler B L. Quorum sensing in bacteria[J]. Annu Rev Microbiol, 2001, 55(1): 165−199.
    [16] Henke J M, Bassler B L. Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi[J]. J Bacteriol, 2004, 186(20): 6902−6914.
    [17] Sun K. Identification and application of a novel genetic marker of Vibrio harveyi[D]. Qingdao: Insti-tute  of  Oceanology  of  the  Chinese  Academy of  Sciences, 2009.孙鲲.哈氏弧菌种特异性分子标记的发现及其在哈氏弧菌快速诊断和免疫防治中的应用[D]. 青岛: 中国科学院海洋研究所, 2009.
    [18] Huang S W, Lu Y Y, Xu D G, et al. Establishment of a re-al-time Taq Man- based PCR method for rapid detection of Vibrio cholerae[J]. Chinese Journal of Health Laboratory Technology, 2006, 16(8): 923−924.黄世旺, 卢亦愚, 徐丹戈, 等. TaqMan荧光定量 PCR 技术快速检测霍乱弧菌方法的建立[J]. 中国卫生检验杂志, 2006, 16(8): 923−924.
    [19] Qin Y Y, Wu H, Xiao X L, et al. Rapid detection of Vibrio parahaemolyticus by TaqMan-based Real-time PCR assay targeting the toxR Gene[J]. Chinese Journal of Biotechnology, 2008, 24(10): 1837−1842.覃倚莹, 吴晖, 肖性龙, 等. toxR基因作为荧光定量 PCR 靶基因设计TaqMan探针快速检测副溶血弧菌 [J]. 生物工程学报, 2008, 24(10): 1837−1842.
    [20] Zhang X J, Chen L, Bi K R, et al. Development of SYBR Green-BasedⅠReal-time quantitative PCR for detection of Vibrio parahaemolyticus[J]. Food Science, 2012, 33(8): 203−206.张晓君, 陈丽, 毕可然, 等. 副溶血弧菌的 SYBR GreenⅠ实时定量 PCR检测方法建立[J]. 食品科学, 2012, 33(8): 203−206.
    [21] Zhang X J, Liang L G, Yan B L, et al. Development of a SYBR green I real-time PCR for detection of Vibrio para-hae-molyticus based on toxR gene[J]. Chinese Journal of Veterinary Science, 2012, 32(4): 543−547.张晓君, 梁利国, 阎斌伦, 等. 基于toxR基因的 SYBR GreenⅠ实时定量 PCR 检测病原副溶血弧菌方法的建立[J]. 中国兽医学报, 2012, 32(4): 543−547.
    [22] Zhang X J, Yao D R, Yan B L, et al. Development of a SYBR green I real-time PCR for detection of Vibrio choierae based on lolB gene[J]. Progress in Fishery Sciences, 2012, 33(2): 104−110.张晓君, 姚东瑞, 阎斌伦, 等. 基于lolB基因的 SYBR Green Ⅰ实时定量 PCR 检测病原霍乱弧菌方法的建立[J]. 渔业科学进展, 2012, 33(2): 104−110.
    [23] Zhou S, Hou Z, Li N, et al. Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyt-icus in seawater and seafood[J]. J Appl Microbiol, 2007, 103(5): 1897−1906.
    [24] Shi X L, Hu Q H, Zhang J F. Rapid simultaneous detection of Salmonella and Shigella using modified molecular beacons and real-time PCR[J]. Chinese Journal of Epidemiology, 2006, 27(12): 1053−1056.石晓路, 扈庆华, 张佳峰. 多重实时 PCR 快速同时检测沙门菌和志贺菌[J]. 中华流行病学杂志, 2006, 27(12): 1053−1056.
    [25] Peng Y Z, Jia H, Zou H, et al. Study on the detection of four intestinal bacteria by using quantitative multi- link real- time PCR[J]. China Tropical Medicine, 2005, 5(5): 943−947.彭雁忠, 贾宏, 邹虹, 等. 多联实时荧光 PCR 定量方法检测四种肠道细菌的研究[J]. 中国热带医学, 2005, 5(5): 943−947.
    [26] Nordstrom J L, Vickery M C L, Blackstone G M, et al. De-velopment of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticusbacteria in oysters[J]. Appl Environ Microbiol, 2007, 73(18): 5840−5847.
    [27] Zhang J, Shi H, Xie J J, et al. Rapid detection of patho-genicVibrio harveyi by loop-mediated isothermal amplifica-tion (LAMP) method[J]. Journal of Shanghai Ocean Uni-versity, 2010, 19(5): 601−607.张静, 施慧, 谢建军, 等. 利用LAMP 法快速检测致病性哈维氏弧菌[J]. 上海海洋大学学报, 2010, 19(5): 601−607.
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廖梅杰,张正,荣小军,王印庚,刘智超,栾晶,李彬,王岚,陈贵平. 基于vhhP2基因的SYBR Green I实时定量PCR检测哈维氏弧菌方法的建立[J]. Jounal of Fishery Sciences of China, 2014,[volume_no](3):611-620

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