| 刘金叶，冯娟，刘广峰，苏友禄，徐力文，郭志勋，王云新.鲨鱼弧菌LAMP检测方法的建立[J].广东农业科学,2012,39(3):146-149 |
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鲨鱼弧菌LAMP检测方法的建立 |
| Rapid detection of Vibrio carchariae by loop-mediated isothermal amplification |
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| DOI： |
| 中文关键词: 鲨鱼弧菌 环介导等温扩增技术 rpoA 基因 |
| 英文关键词: Vibrio carchariae 1oop-mediated isothermal amplification rpoA gene |
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| 摘要点击次数: 1636 |
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| 中文摘要: |
| 采用环介导等温扩增技术（LAMP）成功构建了一种鲨鱼弧菌的检测方法。根据鲨鱼弧菌的rpoA 基因序列设计4条引物（内引物F3 和B3，外引物FIP 和BIP），并对LAMP 扩增反应的各物质浓度和反应条件进行优化，确定最佳反应温度为65℃，最佳反应时间为55 min。该法对鲨鱼弧菌纯菌培养物的检测浓度约为2.4×102 CFU/mL，结果比PCR 法具有相似或更高的灵敏度，而且操作简单，反应后体系变浑浊，肉眼可辨；加入SRYB Gold 荧光染料，阳性反应为绿色，阴性对照为浅橘色，更便于观察，有望发展成为快速检测鲨鱼弧菌的一种有效方法。 |
| 英文摘要: |
| A one step loop-mediated isothermal amplification (LAMP) assay was developed for detection of Vibrio carchariae. A set of four LAMP primers were designed by targeting the rpoA gene and optimized the reaction, finally determinded the reaction at 65℃for 55 min. The detection limit of V. carchariae culture by LAMP was 2.4×102 CFU/mL and the resulrs showed that the LAMP was more sensitivity than PCR. Futhermore, this LAMP method's operation was simple and the result was discernible by eye. If join fluorochrome SRYB Gold, positive reaction for green, negative control for shallow orange, the result was more convenient for observation. So this assay was expected to become a valuable tool for rapid detection and identification of Vibrio carchariae. |
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