Xu Zhenbo, Li Lin, Chu Jin, Peters Brian M, Harris Megan L, Li Bing, Shi Lei, Shirtliff Mark E
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China.
Food Res Int. 2012 Jul 1;47(2):166-173. doi: 10.1016/j.foodres.2011.04.042.
A loop-mediated isothermal amplification (LAMP) method for rapid detection of various Staphylococcus strains and associated antibiotic resistance determinant had been developed and evaluated in this study. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on three targets: 16SrRNA, femA and mecA.. Forty-one reference strains, including various species of gram-negative and -positive isolates, were included in this study to evaluate and optimize LAMP assays. The optimal reaction condition was found to be 65 °C for 45 min, with detection limits at 100 fg DNA/tube and 10 CFU/reaction for 16S rRNA, 100 fg DNA/tube and 10 CFU/reaction for femA, 1 pg DNA/tube and 100 CFU/reaction for mecA, respectively. Application of LAMP assays were performed on 118 various types of Staphylococcus isolates, the detection rate of LAMP assays for the 16SrRNA, femA and mecA was 100% (118/118), 98.5% (64/65) and 94.3% (66/70), and the negative predictive value (NPV) was 100%, 98.1% and 92.3% respectively; with a 100% positive predictive value (PPV) for all three targets. In conclusion, LAMP assays were demonstrated to be useful and powerful tools for rapid detection of various Staphylococcus strains, and undoubtedly, the rapidness, technical simplicity, and cost-effectiveness of LAMP assays will demonstrate broad application for bacteriological detection of food-borne Methicillin-resistant Staphylococcus (MRS) isolates.
本研究开发并评估了一种用于快速检测各种葡萄球菌菌株及相关抗生素耐药决定因素的环介导等温扩增（LAMP）方法。专门设计了六种引物，包括外引物、内引物和环引物，用于识别三个靶标（16SrRNA、femA和mecA）上的八个不同序列。本研究纳入了41株参考菌株，包括各种革兰氏阴性和阳性分离株，以评估和优化LAMP检测方法。发现最佳反应条件为65℃反应45分钟，16S rRNA的检测限分别为100 fg DNA/管和10 CFU/反应，femA为100 fg DNA/管和10 CFU/反应，mecA为1 pg DNA/管和100 CFU/反应。对118株不同类型的葡萄球菌分离株进行了LAMP检测，16SrRNA、femA和mecA的LAMP检测阳性率分别为100%（118/118）、98.5%（64/65）和94.3%（66/70），阴性预测值（NPV）分别为100%、98.1%和92.3%；三个靶标的阳性预测值（PPV）均为100%。总之，LAMP检测被证明是快速检测各种葡萄球菌菌株的有用且强大的工具，毫无疑问，LAMP检测的快速性、技术简便性和成本效益将在食源性耐甲氧西林葡萄球菌（MRS）分离株的细菌学检测中得到广泛应用。
