Developing a real-time quantitative loop-mediated isothermal amplification assay as a rapid and accurate method for detection of Brucellosis
Developing a real-time quantitative loop-mediated isothermal amplification assay as a rapid and accurate method for detection of Brucellosis
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DOI：
10.1111/jam.12290
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发表时间：
2013-09-01
影响因子：
4
通讯作者：
Majidzadeh-A, K.
中科院分区：
文献类型：
--
作者：
Soleimani, M.;Shams, S.;Majidzadeh-A, K.
目的：本研究的目的是设计一种快速检测布鲁氏菌的LAMP方法，并建立一种灵敏的定量LAMP（Q-LAMP）检测布鲁氏菌病的方法。通过制备含有omp 25基因的质粒的连续10倍稀释液，然后进行LAMP反应来评价LAMP方法的灵敏度。为了改进作为定量测试的测定法，通过Loopamp实时浊度计系统评估连续稀释中的LAMP产物，然后通过绘制时间阈值对拷贝数的对数来生成标准曲线。使用布鲁氏菌基因组DNA和含有11种革兰氏阳性和革兰氏阴性微生物基因组的样本组评价了试验专属性。LAMP检测具有高度特异性，在非布鲁氏菌微生物中未观察到扩增产物。目视检测浊度或荧光颜色变化以及琼脂糖凝胶电泳的检测灵敏度分别为560 ng和5.6 ng。结论：本研究建立的LAMP方法是一种简便、快速、灵敏、特异的布鲁氏菌检测方法。本研究的意义和影响：LAMP检测方法由于简单、成本低，在感染性疾病的诊断中可以优先于其他分子方法。
Aim: The aim of this study was designing a LAMP method for the rapid detection of Brucella and development of a sensitive quantitative-LAMP (Q-LAMP) assay for quantification of brucellosis.Methods and Results: In this study for the LAMP detection of the causative agent of brucellosis, we used specifically designed primers to target the omp25 conserved gene of Brucella spp. The sensitivity of the LAMP method was evaluated by preparing serial tenfold dilution of omp25 gene containing plasmid followed by performing the LAMP reaction. To improve the assay as a quantitative test, LAMP products in the serial dilution were evaluated by Loopamp real-time turbidimeter system and then standard curve was generated by plotting time threshold values against log of copy number. The assay specificity was evaluated using Brucella genomic DNA and a panel containing genomes of 11 gram-positive and gram-negative organisms. The LAMP assay was highly specific and no amplification products were observed from the non-Brucella organisms. The test sensitivity for visual detection of turbidity or fluorescent colour change and also agarose gel electrophoresis was 560 ng and 5.6 ng, respectively. The lower limit of detection was 17 copies of the gene that could be detected in 50min.Conclusions: The results of this study indicated that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of Brucella spp. that may improve diagnostic potential in clinical laboratories.Significance and Impact of the Study: The LAMP assay because of the simplicity and low cost can be preferred to other molecular methods in the diagnosis of infectious diseases.