虾夷马粪海胆致病菌强壮弧菌的PCR检测方法

强壮弧菌Vibrio fortis是虾夷马粪海胆Strongylocentrotus intermedius的一种致病菌.为建立强壮弧菌的PCR检测方法,根据强壮弧菌gyrB基因的高度变异区序列设计引物,筛选出特异性强的3对引物VF-6、VF-7及VF-8,分别对强壮弧菌S0907及20株参比菌株进行PCR扩增,结果显示,3对引物均对强壮弧菌扩增出与预期大小一致的目的基因片段且参考菌株无扩增条带.以不同稀释度的菌悬液制备的DNA模板进行PCR扩增,结果显示,3对引物VF-6、VF-7及VF-8可检测的最低细菌浓度分别为4.1×104、4.1×103、4.1 ×102cfu/mL;对不同稀释度的细菌DNA模板进行PCR扩增,结果显示,3对引物VF-6、VF-7及VF-8可检测的最低DNA含量分别为1.4、0.14、0.014 pg/μL.试验结果表明:3对引物的特异性均较好,但灵敏度存在差异,其中以VF-8为引物的PCR检测方法最佳；使用以VF-8为引物的PCR检测方法对实验室养殖海胆及其生境样品进行初步检测,健康海胆、养殖海水及投喂的海带均为阴性,而自然海域海水中带有一定量的强壮弧菌.

PCR detection of pathogen Vibrio fortis in sea urchin Strongylocentrotu intermedius

Vibrio fortis is a pathogenic bacteria of sea urchin Strongylocentrotus intermedius.To establish PCR detection process of V.fortis,three pairs of primer VF-6,VF-7 and VF-8 were designed according to the specified region of gyrB genes of V.fortis.20 strains of bacteria were included in PCR amplification as control.The results showed that the amplified fragments of PCR applied with the three pairs of primer were all agreed with the expected sizes,and no PCR product was found from the other reference strains.Different dilutions of bacterial suspension were used as DNA templates in PCR amplification,the results showed that the three pairs of primer was found to detect the minimum bacterial concentration of 4.1×104,4.1×103,4.1×102 cfu/mL respectively.Meanwhile,different dilutions of DNA were performed in PCR amplification,the results showed that the minimum detectable concentration of bacteria DNA was 1.4,0.14,0.014 pg/μL respectively.These experiments showed that all the three pairs of primers demonstrated excellent specificity to V.fortis.However,the sensitivity of the PCR amplifications was different among the three primers,PCR procedure applied with primer VF-8 showing the best.V.fortis in coelomic fluid of sea urchin and the environmental samples were detected by PCR detection method with VF-8.The results of healthy sea urchin,breeding seawater and feeding kelp were negative,while the sample of seawater from natural sea area had a certain amount of V.fortis.