| 摘要: |
| 针对鰤鱼诺卡氏菌 16S—23S rRNA 基因内转录间隔序列, 设计了四条 LAMP 引物, 在外内引物浓度比 1:8、dNTP 浓度 0.8—1.2mmol/L、Mg2+浓度 10—14mmol/L、反应温度 60—65℃、反应时间 60min 的优化条件下, 扩增产物经电泳后呈现特异性的 LAMP 梯形条带, 建立的 LAMP 法具有高灵敏度, 达到 10-7μg/μl DNA 浓度, 比常规 PCR 方法高 100 倍。将该方法应用于取自养殖场的乌鳢、 大黄鱼、 黄姑鱼组织样品检测, 结果在 16 份组织样品中有 7 份检出自然感染的鰤鱼诺卡氏菌, 与同步取样品鱼组织的细菌分离、 培养检查结果相一致, 并显示既能检测发病鱼, 又能检出未发病且已感染的病鱼。分析表明, 这是一种能快速、简易、特异、敏感的检测鱼类致病鰤鱼诺卡氏菌的诊断方法。
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| 关键词: 鰤鱼诺卡氏菌, 16S—23S rRNA 基因, 环介导恒温基因扩增检测法 |
| DOI：10.11693/hyhz201101004004 |
| 分类号: |
| 基金项目:长江学者和创新团队发展计划资助, IRT0734 号; 农业部公益性行业科研专项, 200903029 号; 浙江省自然科学基金项目, Y3080244 号 |
附件 |
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| DEVELOPMENT AND APPLICATION OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF FISH PATHOGENIC NOCARDIA SERIOLAE |
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WANG Guo-Liang, LIU Lu, XU Yi-Jun
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Key Laboratory of Applied Technology of Marine Biology, Ministry of Education, Faculty of Life Science and Biotechnology, Ningbo University
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| Abstract: |
| To develop a loop-mediated isothermal amplification (LAMP) assay for detection of fish pathogenic Nocardia seriolae. Four primers were designed based on the sequence of the 16S―23S ribosomal RNA internal transcribed spacer region of N. seriolae for the use of LAMP assay. The assay was optimized to the following conditions: 1:8 outer primer concentration ratio, 0.8―1.2mmol/L of dNTP, 10―14mmol/L of Mg2+, at 60―65℃ for 60min. LAMP amplification products had ladder-like bands when electrophoresed on an agarose gel. The assay could amplify up to 10-7μg/μl of DNA. Hence, the sensitivity of LAMP assay was 100-fold higher than the standard PCR protocol. The assay was applied to detect the presence of Nocardia seriolae in different tissue samples from farm Ophiocephalus argus, Pseudnosciaena crocea, Niber albiflora. Seven naturally infected N. seriolae were detected in 16 tissue samples, and the results from the bacterial culture test were consistent with fish tissue samples taken simultaneously. This study showed LAMP assay could be used not only to diagnose the infected fishes, but also to recognize the carrier of N. seriolae as well. LAMP assay is a rapid, simple, specific and sensitive test to diagnose for fish pathogenic N. seriolae.
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| Key words: Nocardia seriolae, 16S―23S rRNA gene, LAMP method |
