Tsai M-A, Wang P-C, Yoshida T, Liaw L-L, Chen S-C
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan, Republic of China.
Dis Aquat Organ. 2013 Feb 28;102(3):225-35. doi: 10.3354/dao02546.
Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.
基于环介导等温扩增（LAMP）鉴定方案，本研究尝试通过使用从加氏乳球菌α/β折叠家族水解酶基因设计的引物对来检测鱼类中的加氏乳球菌。反应时间和温度在60℃下优化为60分钟，通过向反应管中添加SYBR Green I使产生的扩增子可视化。使用45种不同的细菌菌株评估该检测方法的特异性。在来自各种水生动物的所有30株加氏乳球菌分离株中均观察到阳性结果。在15株非加氏乳球菌菌株中未观察到假阳性结果。当使用纯化的加氏乳球菌DNA时，LAMP检测的检测限比靶向16S rDNA的传统聚合酶链反应（PCR）灵敏10倍。使用粗细菌裂解物时，LAMP检测的检测限约为300个菌落形成单位（CFU），比PCR灵敏100倍。此外，使用这种优化的LAMP检测方法检测了实验性攻毒的罗非鱼和鲻鱼的脾脏、肾脏和脑中的加氏乳球菌。本研究结果证明了LAMP在提供一种快速且简单的检测鱼类中加氏乳球菌的方法方面的有效性。
