Establishment and Application of Multiplex PCR Assay for Detection of Three Major Freshwater Pathogens
HU Zongyun, YANG Peimin, YAN Youli
Liaoning Key Laboratory for Prevention and Treatment of Aquatic Animal Diseases, Freshwater Fisheries Research Academy of Liaoning Province, Liaoyang 111000, China
Abstract：A multiplex PCR detection system was developed for three major freshwater pathogens, Aeromonas hydrophila, Edwardsiella tarda and Bacillus cereus, from Liaoning province. hly A, ese D and nhe A were selected for the multiplex PCR. The reaction condition was optimized and the specificity and sensitivity were tested for this method. The results showed that all target fragments were amplified clearly when the concentration of all primer pairs was 0.4 μmol/L, and the concentrations of genomic DNA of A. hydrophila, E. tarda and B. cereus were 2.5, 2.5 and 2.5 ng/μL, as well as dNTP and polymerase were added 2.5 μL and 0.3 μL, respectively, with the annealing temperature of 55 ℃. The sensitivity test showed that the multiplex PCR had a high sensitivity with the detection limit of 4.8 ng for A. hydrophila, 3.0 ng for E. tarda and 2.6 ng for B. cereus. The coincidence rate of the multiplex PCR method and 16S r DNA identification method for suspicious clinical samples was 100%. The established multiplex PCR method was specific, sensitive and rapid to simultaneously detect the three pathogens from freshwater fishes.
胡宗云, 杨培民, 闫有利. 3种淡水鱼病原菌多重PCR检测方法的建立与应用[J]. 水产科学, 2022, 41(4): 605-613.
HU Zongyun, YANG Peimin, YAN Youli. Establishment and Application of Multiplex PCR Assay for Detection of Three Major Freshwater Pathogens. Fisheries Science, 2022, 41(4): 605-613.
[1]HARIKRISHNAN R, RANI M N, BALASUNDARAM C. Hematological and biochemical parameters in common carp, Cyprinus carpio, following herbal treatment for Aeromonas hydrophila infection[J].Aquaculture,2003,221(1/2/3/4):41-50. [2]苏应兵.斑点叉尾鮰暴发性败血症的病原及免疫预防研究[D].武汉:华中农业大学,2006:17-23. [3]PRIDGEON J W, KLESIUS P H, MU X, et al. An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish[J].Journal of Applied Microbiology,2011,111(1):114-124. [4]朱成科,周晓扬,张其中.南方鲇幼鱼细菌性败血症病原与组织病理[J].中国水产科学,2011,18(2):360-370. [5]李楠,郭慧芝,焦冉,等.草鱼的一种急性细菌性传染病病原的分离鉴定及致病性研究[J].水生生物学报,2011,35(6):980-987. [6]梁正生,黄钧,施金谷,等.黄颡鱼腹水病病原菌的分离鉴定及药敏试验[J].南方农业学报,2012,43(9):1400-1404. [7]邓先余,罗文,谭树华,等.黄颡鱼(Pelteobagrus fulvidraco)“红头病”病原菌迟钝爱德华氏菌(Edwardsiella tarda)的分离及鉴定[J].海洋与湖沼,2008,39(5):511-516. [8]耿毅,汪开毓,范方玲,等.养殖黄颡鱼(Pelteobagrus fulvidraco)鮰爱德华氏菌(Edwardsiella ictaluri)的分离鉴定与生物学特性研究[J].海洋与湖沼,2010,41(1):61-67. [9]朱芸.中华鳖“浮肿病”病原菌的鉴定及迟钝爱德华氏菌溶血活化基因的克隆[D].南宁:广西大学,2007:57. [10]陈翠珍.爱德华氏菌及鱼类爱德华氏菌病[J].河北科技师范学院学报,2004,18(3):70-76. [11]王忠敏,黄惠莉.一株罗非鱼出血病致病菌的分离与鉴定及组织病理观察[J].华侨大学学报(自然科学版),2012,33(6):660-666. [12]胡宗云,宋文华,张涛,等.黄颡鱼“红头病”病原菌蜡状芽孢杆菌分离鉴定及药敏分析[J].水产学杂志,2016,29(4):33-37. [13]程俊,杨文涛,袁冬霞,等.一株养殖大菱鲆体表溶血性蜡状芽胞杆菌的分离鉴定及基因组分析[J].基因组学与应用生物学,2016,35(11):3094-3104. [14]BERNARDET J F, CAMPBELL A C, BUSWELL J A. Flexibactermaritimus is the agent of ‘black patch necrosis’ in Dover sole in Scotland[J].Diseases of Aquatic Organisms,1990,8:233-237. [15]PAZOS F, SANTOS Y, MACÍAS A R, et al. Evaluation of media for the successful culture of Flexibactermaritimus[J].Journal of Fish Diseases,1996,19(2):193-197. [16]SAKAI T, YUASA K, SANO M, et al. Identification of Edwardsiella ictaluri and E. tarda by species-specific polymerase chain reaction targeted to the upstream region of the fimbrial gene[J].Journal of Aquatic Animal Health,2009,21(2):124-132. [17]ALTINOK I, CAPKIN E, KAYIS S. Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens[J].Veterinary Microbiology,2008,131(3/4):332-338. [18]钟泽澄,王进,张师音.多重PCR技术研究进展[J].生物工程学报,2020,36(2):171-179. [19]白菊红,康建平,张星灿,等.多重PCR技术在病原微生物检测中的应用[J].食品工业科技,2019,40(7):322-325. [20]秦鑫,王亚东,李红梅,等.基于mtCOⅠ基因的三种短体线虫多重PCR检测[J].农业生物技术学报,2020,28(3):562-570. [21]DEL CERRO A, MARQUEZ I, GUIJARRO J A. Simultaneous detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, three major fish pathogens, by multiplex PCR[J].Applied and Environmental Microbiology,2002,68(10):5177-5180. [22]GONZÁLEZ S F, KRUG M J, NIELSEN M E, et al. Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray[J].Journal of Clinical Microbiology,2004,42(4):1414-1419. [23]李晨,王秀华,黄倢.3种主要水产病原菌多重PCR检测方法的建立[J].渔业科学进展,2010,31(3):100-106. [24]艾克特,徐佩,周静文,等.5种淡水细菌的多重PCR检测方法的建立及其应用[J].水产科学,2015,34(5):321-326. [25]乌日琴,但学明,刘中勇,等.多重RT-PCR体系检测4种虾病毒的方法[J].水产学报,2011,35(3):438-445. [26]刘飞,张宝存,张晓华,等.对虾6种病毒多重PCR检测方法的建立[J].渔业科学进展,2014,35(1):60-67. [27]符贵红,肖丹,胡鲲,等.鲫源嗜水气单胞菌毒力基因多重PCR检测及ERIC-PCR分子分型[J].海洋渔业,2014,36(6):549-556. [28]曾伟伟,王庆,王英英,等.草鱼呼肠孤病毒三重PCR检测方法的建立及其应用[J].中国水产科学,2013,20(2):419-426. [29]侯月娥,伍建敏,王凤求,等.全预混冻干PCR试剂检测草鱼呼肠孤病毒Ⅱ型的研究[J].水产科学,2019,38(1):80-85. [30]叶键,许婷,施礼科,等.3种虾肝肠胞虫PCR检测方法的应用比较分析[J].水产科学,2019,38(3):411-415. [31]饶静静,李寿崧,黄克和,等.致病性嗜水气单胞菌多重PCR检测方法的建立[J].中国水产科学,2007,14(5):749-755. [32]HEUZENROEDER M W, WONG C Y F, FLOWER R L P. Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.:correlation with virulence in a suckling mouse model[J].FEMS Microbiology Letters,1999,174(1):131-136. [33]王斌,赵凤梅,李军伟,等.迟钝爱德华氏菌Ⅲ型分泌系统基因的克隆及多重PCR检测方法的建立[J].大连海洋大学学报,2013,28(3):217-223. [34]TAN Y P, ZHENG J, TUNG S L, et al. Role of type III secretion in Edwardsiella tarda virulence[J].Microbiology (Reading, England),2005,151(Pt 7):2301-2313. [35]XIE G S, HUANG J, ZHANG Q L, et al. A real-time PCR targeted to the upstream regions of HlyB for specific detection of Edwardsiella tarda[J].Chinese Journal of Oceanology and Limnology,2012,30(5):731-737. [36]NGAMWONGSATIT P, BUASRI W, PIANARIYANON P, et al. Broad distribution of enterotoxin genes (hblCDA, nheABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers[J].International Journal of Food Microbiology,2008,121(3):352-356. [37]GUINEBRETIÈRE M H, BROUSSOLLE V, NGU-YEN-THE C. Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains[J].Journal of Clinical Microbiology,2002,40(8):3053-3056. [38]FRICKER M, MESSELHÄUSSER U, BUSCH U, et al. Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks[J].Applied and Environmental Microbiology,2007,73(6):1892-1898. [39]WEHRLE E, MORAVEK M, DIETRICH R, et al. Comparison of multiplex PCR, enzyme immunoassay and cell culture methods for the detection of enterotoxinogenic Bacillus cereus[J].Journal of Microbiological Methods,2009,78(3):265-270. [40]李延利.中华鳖“摇头病”病原的分离鉴定及其对中华鳖4个免疫相关基因的影响研究[D].重庆:西南大学,2020. [41]王琰,高四合,单建杰,等.半滑舌鳎源蜡样芽孢杆菌毒性检测及药敏试验[J].水产科学,2018,37(1):59-65. [42]成翔宇,冯春明,马红丽,等.水产动物主要致病弧菌多重PCR检测方法的建立与应用[J].养殖与饲料,2019(4):22-24. [43]刘礼辉,张德锋,李宁求,等.斑点叉尾鮰败血症3种病原多重PCR检测方法的建立[J].西北农林科技大学学报(自然科学版),2016,44(11):39-46.
2022, Vol. 41 
