Abstract：Abstract Listonella anguillarum has emerged as a serious pathogen causing signi?cant mortalities in various ?sh and shell?sh species over a wide geographical area. With the large-scale application of intensive farming model in aquaculture, the speed of the transmission of aquatic pathogens has been greatly improved. Cultivation of Listonella anguillarum is seen as the gold standard for detection, although it is very time consuming and labour intensive. Accordingly, the establishment of practical and effective diagnostic methods has long been desired. In this study, several pairs of primers and an exo-probe targeting the zinc metalloprotease gene (empA) of Listonella anguillarum was designed and a rapid real-time detection method for Listonella anguillarum based on recombinase polymerase amplification (RPA) was established by optimizing the reaction system and conditions. The results showed that the established Real-time RPA technique could detect Listonella anguillarum directly within 20 min and did not cross-react with other seven common aquatic pathogenic bacteria. The detection limit of Real-time RPA for gDNA of Listonella anguillarum was 1 pg/μL, and reproducibility was good. However, the lowest detection limit of artificial pollution sample was 3.4×102 CFU/mL. It is expected to be the routine which can provide scientific basis and technical reference for the development of rapid diagnostic kit and commercial aquaculture Listonella anguillarum.
[1]Austin B, Austin D A.Bacterial fish pathogens: disease in farmed and wild fish.[M] Bacterial fish pathogens : disease in farmed and wild fish. E. Horwood, Halsted Press, 2012:77-79.[2]Iwamoto M, Ayers T, Mahon B E, et al.Epidemiology of Seafood-Associated Infections in the United States[J].Clinical Microbiology Reviews, 2010, 23(2):399-410[3]Nishibuchi M, Muroga K, Seidler R J, et al.Pathogenic Vibrio isolated from cultured eelsIV. Desoxyribo nucleic acid studies.[J].Nsugaf, 1979, 45(12):1469-1473[4]Bricknell I R, Bowden T J, Verner-Jeffreys D W, et al.Susceptibility of juvenile and sub-adult Atlantic halibut (Hippoglos sushippoglossusL) to infection by Vibrio anguillarum and efficacy of protection induced by vaccination[J].Fish & Shellfish Immunology, 2000, 10(4):319-327[5]李清禄，陈强.海水网箱养殖大黄鱼细菌性病原鉴定与感染治疗研究[J].应用与环境生物学报, 2001, 7(5):489-493[6]张晓君，秦国民，阎斌伦，等.半滑舌鳎病原鳗利斯顿氏菌表型及分子特征研究[J].海洋学报, 2009, 31(5):112-122[7]Walker G T, Fraiser M S, Schram J L, et al.Strand displacement amplification--an isothermal,in vitro DNA amplification technique[J].Nucleic Acids Research, 1992, 20(7):1691-6[8]Notomi T, Okayama H, Masubuchi H, et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research, 2000, 28(12):E63-E63[9]Piepenburg O, Williams C H, Stemple D L, et al.DNA Detection Using Recombination Proteins[J].PLoS biology, 2006, 4(7):1115-1121[10]Milton D L, Norqvist A, Wolfwatz H.Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum[J].Journal of Bacteriology, 1992, 174(22):7235-44[11]Hickey M E, Richards G P, Lee J L.Development of a two-step,non-probed multiplex real-time PCR for surveilling Vibrio anguillarum in seawater[J].Journal of Fish Diseases, 2015, 38(6):551-559[12]Crannell Z A, Rohrman B, Richardskortum R.Quantification of HIV-1 DNA using real-time recombinase polymerase amplification[J].Analytical Chemistry, 2014, 86(12):5615-5623[13]Abd E W A, Patel P, Faye O, et al.Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection[J].Plos One, 2015, 10(6):2234-2238[14]Faye O, Faye O, Soropogui B, et al.Mobile deployment of recombinase polymerase amplification based rapid diagnostics for Ebola virus disease in Guinea in 2015[J].Journal of Clinical Virology, 2015, 70(6):S13-S13[15]Gao W, Huang H, Zhang Y, et al.Recombinase Polymerase Amplification-Based Assay for Rapid Detection of Listeria monocytogenes,in Food Samples[J].Food Analytical Methods, 2016, 10(6):1-10[16]Tang Y, Cao Y, Yu Y, et al.Real-Time Recombinase Polymerase Amplification Assay for the Detection of Vibrio cholerae, in Seafood[J].Food Analytical Methods, 2017, 10(1):1-10[17]徐潮，李亮，金芜军，等.荧光技术检测转基因水稻科丰号[J].分子植物育种, 2014, 12(5):875-880[18]Xia X, Yu Y, Weidmann M, et al.Rapid Detection of Shrimp White Spot Syndrome Virus by Real Time,Isothermal Recombinase Polymerase Amplification Assay[J].Plos One, 2014, 9(8):e104667-e104667[19]Xia X, Yu Y, Hu L, et al.Rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) by real-time,isothermal recombinase polymerase amplification assay[J].Archives of Virology, 2015, 160(4):987-994[20]余俊红，陈吉祥，厉云，等.聚合酶链反应检测花鲈鳗弧菌感染[J].海洋科学进展, 2002, 20(2):60-64[21]Rodkhum C, Hirono I, Crosa J H, et al.Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum[J].Journal of Microbiological Methods, 2006, 65(3):612-618[22]Hong G E, Kim D G, Bae J Y, et al.Species-specific PCR detection of the fish pathogen,Vibrio anguillarum,using the amiB gene,which encodes N-acetylmuramoyl-l-alanine amidase[J].Fems Microbiology Letters, 2007, 269(2):201-6[23]Xiao P, Mo Z L, Mao Y X, et al.Detection of Vibrio anguillarum by PCR amplification of the empAgene[J].Journal of Fish Diseases, 2009, 32(3):293-296[24]董培培，李长红，丁文超，等.环介导等温扩增技术与横向流动试纸条法快速检测鳗利斯顿氏菌的研究[J].水产科学, 2011, 30(2):63-68[25]梁君妮，尹伟力，刘宁，等.鱼类鳗弧菌赖解旋酶基因扩增技术的建立[J].中国预防兽医学报, 2015, 37(8):628-631[26]耿伟光，史成银，李晋，等.同步检测海水养殖动物种病原菌的扩增子拯救多重-方法的建立与应用[J].农业生物技术学报, 2013, 21(9):1125-1134[27]Rodkhum C, Hirono I, Crosa J H, et al.Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum[J].Journal of Microbiological Methods, 2006, 65(3):612-618
