哈维氏弧菌毒力相关基因的筛查及快速检测技术研究-遗传学专业论文.docxVIP

哈维氏弧菌毒力相关基因的筛查及快速检测技术研究不清楚。此前仅知道哈维氏弧菌toxR基因的部分序列，因此克隆toxR基因的 哈维氏弧菌毒力相关基因的筛查及快速检测技术研究 不清楚。此前仅知道哈维氏弧菌toxR基因的部分序列，因此克隆toxR基因的 全长，成为下‘步研究的首要条件。本文利用LAPCR试剂盒，设计‘内外两对引物， 根据试剂盒的操作步骤，通过连续两次PCR，成功地克隆了哈维氏弧菌toxR基 因的部分未知序列，并进行了序列测定。 toxS,tcpA，zDt力船和flaC是霍乱弧菌和副溶血弧菌的重要毒力基因。 本文根据GenBank提供的序列设计引物，通过PCR检测它们在哈维氏弧菌中的分 布情况。结果显示，toxS基因在25株弧菌标准菌株(2株哈维氏弧菌的结果为 阴性)中，有8株PCR扩增获得特异性片段，其它哈维氏弧菌中有4株结果为阳 性。zot基因在25株弧菌标准菌株(2株哈维氏弧菌的结果为阴性)中有2株获 得目的片段，其它哈维氏弧菌结果为阴性。tcpA在25株弧菌标准菌株(2株哈 维氏弧菌的结果为阴性)中有4株结果为阳性，其它哈维氏弧菌结果为阴性。门却 在25株弧菌标准菌株(2株哈维氏弧菌的结果为阳性)有11株结果为阳性，其 它哈维氏弧菌28株结果为阳性。f]aC在25株弧菌标准菌株中(2株哈维氏弧菌 的结果为阴性)有13株结果为阳性，其它哈维氏弧菌中12株结果为阳性。将不 同的哈维氏弧菌菌株注射斑马鱼，对这五种基因与哈维氏弧菌的致病性的关系作 了研究，结果显示这五种基因的分布与哈维氏弧菌致病性没有明显的相关性。 关键词：哈维氏弧菌，f“B快速检测，毒力基因 11 呤维氏弧菌毒力相关基囡的筛查及快速检测技术研究Screening 呤维氏弧菌毒力相关基囡的筛查及快速检测技术研究 Screening of virulence genes and establishment of rapid identification method of l握brio harveyi Abstract Fibrio harveyi is Gram—negative，luminous bacterium and widly distributed in aquatic environment．It is one of the most important pathogenic agents for aquatic animals．It is important to establish a rapid，specific method of detecting矿harveyi The virulence factors of矿harveyi that have been found were extracellular hemolysin， proteases(including metalloprotease and serine protease)，phospholipases and lipases． Whether there are other virulence genes are not clear．In this study,five virulence—related genes to other l,qbrio species were chosen，and their distribution in矿 harveyi WaS detected． toxR(toxin regulator)is widly distributed in Hbrio species，which WaS discovered as a positive transcriptional regulator of many virulence genes in Vibrio species，and the homology is low in different l,qbrio species．In this study．a pair of specific primers based on toxR gene were designed，and it was used to detect矿harveyi by PCR amplification from 56 Fibrio reference strains．All the 20 strains of矿harveyi (including two type strains of矿harveyi)could amply a 382 bp fragment．The detecting percent is 1 00％．The PCR amplification targeting the toxR