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Position: Home > Articles > 半滑舌鳎创伤弧菌分离鉴定及其荧光定量PCR方法的建立 Journal of Fisheries of China 2019 (5)

半滑舌鳎创伤弧菌分离鉴定及其荧光定量PCR方法的建立

作  者：
胡秀彩;王英杰;吕爱军;孙敬锋;王庆奎;廉正义;刘小雪;宋亚娇
单  位：
天津农学院水产学院;天津农学院水产学院天津市水产生态及养殖重点实验室;河南师范大学水产学院
关键词：
半滑舌鳎;创伤弧菌;组织病理;荧光定量PCR
摘  要：
为确定患病半滑舌鳎的病原,从其体内分离到3株革兰氏阴性菌,将其分别命名为ST-1、ST-3和ST-6,进行细菌理化特性、药敏试验、16S rDNA测序以及组织病理学观察等方面的研究,并建立了外膜蛋白(OMP)基因的SYBR Green I荧光定量PCR检测方法.结果显示,3株分离菌株的理化特性符合创伤弧菌特征,16S rDNA与创伤弧菌标准菌株ATCC29307序列相似性达99%以上,系统发育树分析显示,3株分离株与创伤弧菌自然聚为一支,最终鉴定为创伤弧菌;药敏试验显示,3株菌对头孢克肟、头孢哌酮、链霉素、亚胺培南和氟苯尼考等药物高度敏感.人工回归感染试验表明,分离株具有致病性,且半滑舌鳎、斑马鱼的临床症状与自然患病鱼症状相似;组织病理学观察显示,鳃丝上皮细胞增生,鳃小片肿胀黏连、颗粒细胞和黏液细胞增多,肝脏中央静脉淤血,肾脏出血、坏死,肠道黏膜固有层萎缩、黏液细胞增多等.进一步建立OMP基因的荧光定量PCR方法,结果显示,标准曲线的相关系数为0.995,检测下限为1.88*10~2 CFU/mL.以上结果可为半滑舌鳎弧菌病的致病机理、分子诊断和防治提供科学参考.
作  者：
HU Xiucai;WANG Yingjie;Lü Aijun;SUN Jingfeng;WANG Qingkui;LIAN Zhengyi;LIU Xiaoxue;SONG Yajiao;Tianjin Key Lab of Aqua-Ecology and Aquaculture, College of Fisheries, Tianjin Agricultural University;College of Fisheries, Henan Normal University;
单  位：
HU Xiucai%WANG Yingjie%Lü Aijun%SUN Jingfeng%WANG Qingkui%LIAN Zhengyi%LIU Xiaoxue%SONG Yajiao%Tianjin Key Lab of Aqua-Ecology and Aquaculture, College of Fisheries, Tianjin Agricultural University%College of Fisheries, Henan Normal University
关键词：
Cynoglossus semilaevis;;Vibrio vulnificus;;histopathology;;fluorescence quantitative PCR
摘  要：
In order to investigate the pathogen of the diseased tongue sole Cynoglossus semilaevis, three strains of Gram-negative bacteria isolated from diseased C. semilaevis were tentatively named strains ST-1, ST-3 and ST-6,which were identified by physiological and biochemical characteristics, antimicrobial susceptibility test, 16 S rDNA gene sequences analysis and histopathology examination. Furthermore, the SYBR Green quantitative real-time PCR(qRT-PCR) targeting the outer membrance protein(OMP) gene was established. The biochemical results of the isolate ST-1, ST-3 and ST-6 were consistent with the properties of V. vulnificus. The BLAST alignments of the16 S rDNA gene sequence showed that the isolates ST-1, ST-3 and ST-6 shared the identities above 99% with type strain Vibrio vulnificus ATCC 29307(X74727). The phylogenetic tree indicated the isolates clustered with V.vulnificus ATCC 29307. The isolates were eventually identified as V. vulnificus. Antimicrobial susceptibility testing showed that the isolates were highly susceptible to cefixime, cefoperazone, streptomycin, imipenem and florfenicol. The challenge experiments showed that the isolated strains were pathogenic on C. semilaevis and zebrafish(Danio rerio), and similar symptoms were observed with the natural infection. Histopathology examination revealed the hyperplasia of the epithelial cells in the primary lamella, the swelling of the secondary lamella and the increase of the number of granulosa cells and mucus cells, hepatic sinusoid and the congestion of central vein in the liver, hemorrhagic-necrotic lesion in the kidney, the atrophy of the lamina propria and the increase of the number of mucus cells in the intestine. The SYBR Green I quantitative PCR(qPCR) method based on the OMP gene was further established and the results showed that the correlation coefficient of standard curve was 0.995 and the lowest limit of detection was 1.88×102 CFU/mL. The results of this study will lay a foundation for the pathogenesis and prevention of C. semilaevis vibriosis, which can provide scienti?c reference for the molecular diagnosis of the disease.

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