检测鲆鱼腹水病病原菌迟缓爱德华氏菌和溶藻弧菌的嵌套PCR方法
DETECTION OF PATHOGENIC BACTERIAL (EDWARDSIELLA TARDA AND VIBRIO ALGINOLYTICUS) ASSOCIATED WITH SWOLLEN ABDOMEN OF CULTURED TURBOT (SCOPHTHALMUS MAXIMUS) AND FLOUNDER (PARALICHTHYS OLIVACEUS) BY NESTED-PCR
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摘要: 本文首次建立了用嵌套式聚合酶链式反应(Nested-PCR)分别检测天津地区海水工厂化养殖大菱鲆和褐牙鲆腹水病病原菌迟缓爱德华氏菌(Edwardsiella tarda)和溶藻弧菌(Vibrio alginolyticus)的方法。以E781F和E781R为引物,扩增迟缓爱德华氏菌溶血素基因中781bp的片段,再以E485F和E485R为引物,扩增其中的485bp的片段;以V899F和V899R为引物,扩增溶藻弧菌胶原酶基因中899bp片段,再以V550F和V550R为引物,扩增其中的550bp片段。以其他常见海水鱼类致病菌为阴性对照,结果均无非特异性扩增。对迟缓爱德华氏菌和溶藻弧菌的检出灵敏度分别可达10fg和1fg细菌DNA,显著优于目前的检测方法。作者应用该技术对天津地区海水工厂化大菱鲆和褐牙鲆于2006年3月至10月连续进行监测,为有效地控制该病的流行提供了技术支撑。Abstract: Turbot(Scophthatmus maximus) and flounder(Paralichthys olivaceus) are rare and economic fish in the north of China.In recent years,they have become the dominant cultured varieties.Tianjin is the main cultural area of turbot and flounder.However,the prevalence of diseases of the cultured turbot and flounder has resulted in great loss to the cultural enterprise.Swollen abdomen disease was the serious threat to the turbot and flounder according to the investigation.The mortality can reach 80%-90% while the disease broke out.On the foundation of the former work,we have confirmed that Edwardsiella tarda and Vibrio alginolyticus were the pathogenic bacterial associated with swollen abdomen disease.E.tarda is an important pathogenic bacteria to the maricultured fish,furthermore,it can cause other diseases to birds,amphibian and mammals.V.alginolyticus is an opportunist pathogen,which exists extensively in seawater,and causes serious diseases to maricultured fish and even human being.The treatment of the aquatic animals' bacterial disease mostly depend on the antibiotics recently.The misuses of the antibiotics have given more pressures to the human's health.Meanwhile,the antibiotics have brought more opportunities to the pathogenic bacterial to adapt it.Hence,it is necessary to develop a sensitive and reliable method to detect the pathogenic bacterial.We expect to build a system which can forecast the prevalence of the swollen abdomen disease through some detecting methods.In this paper,a diagnostic method was first developed.Two pairs of primers,E781F and E781R,E485F and E485R,were designed for amplification of 781 bp and 485 bp DNA fragments of the hemolytic gene of E.tarda respectively.Similarly,two pairs of primers,V899F and V899R,V550F and V550R,were designed for amplification of 899 bp and 550 bp DNA fragments of the collagenase gene of V.alginolyticus respectively.No specific fragments were amplified when other principal oceanic fish bacterial pathogens were used as negative controls.The sensitivity of the detection of E.tarda and V.alginolyticus was 10fg and 1fg respectively,which are better than the other diagnostic methods.Nested-PCR detected the presence of E.tarda and V.alginolyticus in 32 samples which were the cultured turbot and flounder taken from the farms of Tianjin,23(23/32) of which were positive for E.tarda,11(11/32) of which were positive for V.alginolyticus,and 2(2/32) of which were positive for both organisms.The results showed that the nested-PCR was effective and reliable methods for the detection of E.tarda and V.alginolyticus.
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Keywords:
- Swollen Abdomen /
- Edwardsiella tarda /
- Vibrio alginolyticus /
- PCR /
- Detectingm ethods
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[1] Xu e S X, Feng SM, Sun J S. Isolat ion and iden t if icat ion of patho??gen ic bacteria associated w ith sw ollen abdom en of cu ltured tu rbot(S coph thalmu s maximu s) and f lounder ( Pa ral ich thys olivaceu s) [J]. Oceanologia E t L im nolog ia S in ica, 2006, 37: 548-554 [薛淑霞, 冯守明, 孙金生. 海水工厂化养殖大菱鲆( S coph thalmu smaximu s)和褐牙鲆(P ara lich thys oliva ceu s)腹水病病原菌的分离与鉴定. 海洋与湖沼, 2006, 37: 548-554]
[2] W h ite F H, S im pson C F, W illiam s L E, et al. Isolat ion ofE d??wa rd siella tarda from aquat ic an im al sp ecies and surface w atersin Florida [J]. Jou rnal of W ild lif e D isea se, 1973, 9 ( 3 ) :204-208
[3] Bragg R R. First iso lation ofEdw ard sie lla tarda from f ish in SouthA frica [J]. Bu lletin of the Eu ropean Associa tion of F ish P atholo??g ists, 1998, 8 ( 4) : 87-88
[4] Janda JM, Abbott S L. In fect ion s associated w ith the genu s Ed??w ard siella: the ro le ofEdw ard sie lla tarda in hum an d isease [J].C lin ica l Inf ectious D isease, 1993, 17 ( 4) : 742-748
[5] S elvin J, L inpton A P. Vibrio a lg inolyticu s associated w ith wh itespot d isease ofP enaeus m onodon [J]. D is. Aqua t. Organ., 2003,57: 147-150
[6] X ie Z, H u C, Chen C, et a l. In vestigation of sevenV ib rio viru len cegenes am ong Vibrio a lg in olyticu s and Vibrio parahaenolyticusstrains from the coastalM ari culture system s in Gu angdong, C hina [J]. L et ters in Applied M icrobiology, 2005, 41: 202-207
[7] L iu P C, L in JY, H aiao P T, et a l. Isolation and characterizationof pathogen ic Vibrio alginolyticus from d isease ofP enaeu s mon??od on [J]. D is. A quat. Organ., 2003, 57: 147-150
[8] Francis P, Kheng L, H en ry H. Vibrio a lg inoly ticu s in fect ion s inH aw aii [J]. C l iln ica lM icrobiology, 1977, 5( 6 ): 670-672
[9] Zuo F Q, J ian JC, W u ZH. Characterization of ex tracellu lar p rod??ucts from Vibrio a lg in olyticu s isolated from m aricu ltured fish [J].A ctaH yd robiolog ica S inica, 2006, 30( 5 ): 553-558 [左凤琴, 简纪常, 吴灶和. 鱼源溶藻弧菌胞外产物的特性研究. 水生生物学报, 2006, 30( 5) : 553-558]
[10] L iu H, Gao L Y, Sh iX J. Detection ofR enibac terium salm onina??rum by n ested??PCR [J]. Jou rnal of F isheries of Ch ina, 2002, 26:453-458 [刘荭, 高隆英, 史秀杰. 用nested??PCR快速检测鲑鱼肾杆菌. 水产学报, 2002, 26: 453-458]
[11] H iroak iT, Yu j i S, Kazuyuk iM, et a l. Stru ctura l gene and com??p lete am ino acid sequen ce of Vibrio alg inoly ticus collagenase [J]. B iochem, 1992, 281: 703-708
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