细鳞鱼温和气单胞菌LAMP检测方法的建立

在对野生细鳞鱼保护和驯养繁育工作中发现其出现厌食独居、水中打转、体表溃疡和肝肾脏出血症状,从患病鱼体内分离得到1株细菌,经过生理生化鉴定和16S rRNA测序确定该菌株为温和气单胞菌(Aeromonas sobria),在此基础上利用环介导恒温扩增技术(LAMP)建立该菌的快速检测方法。选用管家基因zipA作为靶基因,设计筛选出一套最佳引物,使用25μL的优化体系,提取几种常见水产致病菌株DNA进行特异性分析,以10倍稀释法进行LAMP灵敏性试验,确定最低检测DNA浓度为1.663×10^-9 ng/μL。试验表明建立的温和气单胞菌LAMP检测方法可及早发现感染细菌,为阻止细鳞鱼疫情暴发和细鳞鱼驯化带来保障。

Establishment of a LAMP method for detecting Aeromonas sobria in Brachymystax lenok

In the protection and domestication and breeding of the squid,it has been found that the squid often suffer from anorexia,turbulence in water,surface ulcers,and liver and kidney bleeding.For early diagnosis and early treatment of these diseases,one bacterial strain was isolated from the diseased fish.Based on the isolation,physiological and biochemical identification and 16 S rRNA sequencing,the strain was identified as Aeromonas sobria.Then,loop-mediated isothermal amplification(LAMP)was used to establish a method for rapid detection of this bacterium.Furthermore,the housekeeping zipA gene of the strain was selected as the target gene,and a set of optimal primers was designed and selected.Next,using an optimized system of 25μL,the DNA of several common aquatic pathogenic strains was extracted for specific analysis,and was diluted by 10-fold.The LAMP sensitivity test was performed to determine the minimum detection DNA concentration to be 1.663×10^-9 ng/μL.The results showed that this method can be used to detect infectious A.sobria early and in time,facilitating prevention of the outbreak of the disease and effective domestication of the squib.