Development and application of loop-mediated isothermal amplification assays on rapid detection of various types of staphylococci strains.
Development and application of loop-mediated isothermal amplification assays on rapid detection of various types of staphylococci strains.
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DOI：
10.1016/j.foodres.2011.04.042
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发表时间：
2012-07-01
影响因子：
8.1
通讯作者：
Shirtliff, Mark E.
中科院分区：
文献类型：
--
作者：
Xu, Zhenbo;Li, Lin;Chu, Jin;Peters, Brian M.;Harris, Megan L.;Li, Bing;Shi, Lei;Shirtliff, Mark E.
建立了一种快速检测葡萄球菌及其耐药基因的环介导等温扩增（LAMP）方法。设计了6条引物，包括外引物、内引物和环引物，用于识别16 SrRNA、femA和mecA 3个靶点上的8个不同序列。本研究包括41种参考菌株，包括各种革兰氏阴性和阳性分离株，以评价和优化LAMP检测。发现最佳反应条件为65 °C 45 min，16 S rRNA的检测限为100 fg DNA/管和10 CFU/反应，femA的检测限为100 fg DNA/管和10 CFU/反应，mecA的检测限为1 pg DNA/管和100 CFU/反应。对118株不同类型葡萄球菌进行LAMP检测，16 SrRNA、femA和mecA的检出率分别为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%;所有三个靶点的阳性预测值（PPV）均为100%。总之，LAMP检测被证明是有用的和强大的工具，用于快速检测各种葡萄球菌菌株，毫无疑问，快速，技术简单，成本效益的LAMP检测将证明广泛的应用于细菌学检测食源性耐甲氧西林葡萄球菌（MRS）分离株。
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.
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DOI：
10.1016/j.ijfoodmicro.2008.12.007
发表时间：
2009-08-31
影响因子：
5.4
作者：
de Boer, E.;Zwartkruis-Nahuis, J. T. M.;Heuvelink, A. E.
通讯作者：
Heuvelink, A. E.
影响因子：
3.3
作者：
Khanna, T.;Friendship, R.;Weese, J. S.
通讯作者：
Weese, J. S.
影响因子：
3.3
作者：
Nagamine, K;Hase, T;Notomi, T
通讯作者：
Notomi, T
影响因子：
--
作者：
Kaneko, Hisatoshi;Kawana, Takashi;Suzutani, Tatsuo
通讯作者：
Suzutani, Tatsuo
影响因子：
9.4
作者：
Shimizu, A;Fujita, V;Kawano, J
通讯作者：
Kawano, J